Identification of antibiotic resistance using labelled antibiotics

ABSTRACT

Subject of the present invention is a method for detection of an antibiotic resistance in a micro-organism comprising the steps of exposing suspected micro-organism to a labelled (fluorescent) antibiotic and observing the differences between it and a non-resistant micro-organism of the same type.

Subject of the present invention is a method for detection of anantibiotic resistance in a micro-organism.

The characterisation of micro-organisms in routine diagnostic proceduresencompasses the determination of a species' identity and its sensitivitytowards antibiotics. In order to achieve this, micro-organisms need tobe taken from their environment and enriched in a selective environmentfor the separate identification (ID) and antibiotic sensitivity testing(AST). Currently the AST/ID of micro-organisms is achieved byidentifying presence or absence of an array of biochemical features andthe (non-) capability to grow in the presence of antibiotics.Alternatively DNA can be extracted from a sample and the then pooled DNAis tested for the presence/absence of specific sequences utilising geneamplification techniques. This can signal the presence of an organism inthe sample. Equally, the presence of a gene coding for antibioticresistance in the sample can be detected. By definition, extracting DNAdirectly from a sample renders pooled DNA from an unknown mixture ofcells. Unequivocal results can only be achieved if the DNA is extractedfrom a pure colony.

Staphylococcus aureus is one of the most common causes of nosocomial orcommunity-based infections, leading to serious illnesses with high ratesof morbidity and mortality. In recent years, the increase in the numberof bacterial strains that show resistance to methicillin-resistantStaphylococcus aureus (MRSA) has become a serious clinical andepidemiological problem because this antibiotic (or analogue) isconsidered the first option in the treatment of staphylococciinfections. The resistance to this antibiotic implies resistance to allβ-lactam antibiotics. For these reasons, accuracy and promptness in thedetection of methicillin resistance is of key importance to ensurecorrect antibiotic treatment in infected patients as well as control ofMRSA isolates in hospital environments, to avoid them spreading.

MRSA strains harbour the mecA gene, which encodes a modified PBP2protein (PBP2′ or PBP2a) with low affinity for methicillin and allβ-lactam antibiotics. Phenotypic expression of methicillin resistancemay alter depending on the growth conditions for S. aureus, such astemperature or osmolarity of the medium, and this may affect theaccuracy of the methods used to detect methicillin resistance (1).Hetero-resistant bacterial strains may evolve into fully resistantstrains and therefore be selected in those patients receiving β-lactamantibiotics, thus causing therapeutic failure. From a clinical point ofview they should, therefore, be considered fully resistant.

There are several methods for detecting methicillin resistance (1,9)including classical methods for determining a minimum inhibitoryconcentration MIC (disc diffusion, Etest, or broth dilution), screeningtechniques with solid culture medium containing oxacillin, and methodsthat detect the mecA gene or its protein product (PBP2′ protein) (3,4).Detection of the mecA gene is considered as the reference method fordetermining resistance to methicillin (1). However, many laboratoriesthroughout the world do not have the funds required, the capacity or theexperienced staff required to provide molecular assays for detectingMRSA isolates. It is therefore essential that other, more useful,screening methods are incorporated into routine clinical practice.Moreover, the presence of antibiotic resistance has it's relevance atseveral levels, all of which are of clinical significance

-   1. Presence of a gene conveying resistance, such as mecA, mef(E),-   2. Presence of a repressor gene inhibiting the phenotypic expression    of said resistance mechanism; e.g. MecA repressor-   3. Multiple resistance mechanisms; e.g. Macrolide resistance via    modification of the ribosomal binding site and presence of efflux    mechanism(s).-   4. Level of expression of said resistance mechanism regulated via    transcription and translation detectable as the phenotype

Current cultural techniques require the isolation of a discrete colonyand the subsequent identification and resistance testing, assuming thata single colony is derived from a single cell and is therefore deemed tobe pure. In reality however, the generation of a pure colony from aclinical sample, where pathogens frequently live in bio-filmcommunities, cannot be guaranteed. Equally, using amplificationtechnologies, nucleic acid sequences from multiple cells are extractedand amplified and can therefore render false positive results. Only ifidentification and resistance can be performed and be read on individualcells, is it possible to a true picture of the invading pathogen.

A wide range of antibiotics carry a primary amino group. It is wellknown in the art that reagents such as Fluoresceinisothiocyanate (FITC),Fluorecein-N-hydroxysuccinimide ester will readily react with saidprimary amines.

The increasing spread of antibiotic resistance in both community andhealthcare systems necessitates the precision and speed of molecularbiology. However, the complexity and cost of these assays prohibits thewidespread application in a routine testing environment.

Taking into account the difficulties in identifying a micro-organism andits potential resistance against an antibiotic in a biological sample,it is desirable to be able to quickly identify a pathogen directly froma sample without culturing and without amplification and in addition tobe able to detect or exclude the presence of resistance towards anantibiotic of choice.

It is the intention of this invention to provide a solution by enablingthe simultaneous identification and resistance testing on the cellularlevel. This reduces the complexity of the assays so that an unambiguousassignment of a phenotype can be made for individual cells. The assaysare designed to reduce handling and turnaround time to enable screeningprogrammes such as the screening of all incoming patients for e.g. MRSA.

A first subject of the present invention is thus a method for thedetection of an antibiotic resistance in a predetermined micro-organismin a biological sample, comprising the steps:

-   -   (a) providing a labelled antibiotic,    -   (b) contacting the labelled antibiotic with a biological sample        comprising the micro-organism under conditions which allow        binding of the labeled antibiotic to its binding site in the        micro-organism,    -   (c) detecting the labelled antibiotic in the micro-organism, and    -   (d) identifying a micro-organism in which the amount of        detectable label is altered with respect to the amount of        detectable label in the predetermined micro-organism in its        non-resistant form,        wherein microorganisms identified in step (d) are microorganism        resistant against the antibiotic.

The underlying principle of the method is that if an organism issensitive or resistant to an antibiotic, it will markedly differ fromits resistant or sensitive counterpart. The antibiotics may bind totheir respective binding sites either in the cell lumen, cytoplasm, thecell wall or to secreted proteins such as beta-lactamases. Dependingupon the resistance mechanism, resistant organisms may mostly showeither reduced or no affinity to the antibiotic due to reduced affinityto e.g. ribosomes or penicillin binding proteins. Conversely, if theresistance mechanism is due to the ab/adsorbtion to the outer cellmembrane, the resistant organism will exhibit highly enhancedfluorescence.

The biological sample may be any sample of biological origin, such as aclinical or food sample, suspected of comprising an antibiotic-resistantmicroorganism. The micro-organism may be selected from the groupconsisting of bacteria, yeasts and moulds, in particular from Grampositive and Gram negative bacteria.

Preferably, the predetermined micro-organism is selected from the groupconsisting of Staphylococcus, Enterococcus, and Streptococcus.

More preferably, the predetermined micro-organism is selected from thegroup consisting of Methicillin resistant Staphylococcus, Vancomycinresistant Staphylococcus, Vancomycin resistant Enterococcus, and highlevel Aminoglycoside resistant Enterococci.

The microroganism is even more preferably selected from the groupconsisting of Staphylococcus aureus, Methicillin ResistantStaphylococcus aureus (MRSA), Vancomycin Resistant Staphylococcus aureus(VRSA), Vancomycin Resistant Staphylococcus (VRS), Vancomycin ResistantEnterococci (VRE), Streptococcus pneumoniae, drug resistantStreptococcus pneumoniae (DRSP), and Aminoglycoside resistantEnterococci (HLAR).

The antibiotic to be provided in step (a) may be any antibiotic.Preferably, the antibiotic is selected from the group consisting ofaminoglycosides, carbacephems, carbapenems, cephalosporins,glycopeptides, macrolides, monobactams, beta-lactam antibiotics,quinolones, bacitracin, sulfonamides, tetracyclines, streptogramines,chloramphenicol, clindamycin, and lincosamide.

More preferably, the antibiotics are selected from beta-lactamantibiotics, macrolides, lincosamide, and streptogramins.

Even more preferably, the antibiotic is selected from the groupconsisting of Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin,Streptomycin, Tobramycin, Loracarbef, Ertapenem, Imipenem, Cilastatin,Meropenem, Cefadroxil, Cefazolin, Cephalexin, Cefaclor, Cefamandole,Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren,Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten,Ceftizoxime, Ceftriaxone, Cefsulodine, Cefepime, Teicoplanin,Vancomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin,Roxithromycin, Troleandomycin, Aztreonam, Amoxicillin, Ampicillin,Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin,Mezlocillin, Nafcillin, Penicillin, Piperacillin, Ticarcillin,Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin,Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin,Ofloxacin, Trovafloxacin, Mafenide, Prontosil, Sulfacetamide,Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,Trimethoprim, Trimethoprim sulfa, Sulfamethoxazole, Co-trimoxazole,Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline,Chloramphenicol, Clindamycin, Ethambutol, Fosfomycin, Furazolidone,Isoniazid, Linezolid, Metronidazole, Mupirocin, Nitrofurantoin,Platensimycin, Pyrazinamide, Quinupristin/Dalfopristin, Rifampin,Spectinomycin, Amphotericin B, Flucanazole, Fluoropyrimidins,Gentamycin, and clavulanic acid.

Most preferably, the antibiotic is selected from Vancomycin,Methicillin, Clindamycin, Trimethoprim, Trimethoprim sulfa, Gentamycin,and clavulanic acid.

Surprisingly, a modification of an antibiotic with a labelling groupdoes not hinder the binding of an antibiotic to its binding site in themicro-organism.

Preferably, the antibiotic is labelled by a luminescent labelling group.Many fluorophores suitable as labelling groups in the present inventionare available. The labelling group may be selected to fit the filterspresent in the market. The antibiotic may be labelled by any suitablelabelling group which can be detected in a micro-organism. Preferably,the labelling group is a fluorescent labelling group. More preferably,the labelling group is selected from Fluorescein and Atto-495-NSI.

The labelling group may be coupled to the antibiotic at a functionalgroup. A wide range of antibiotics carry a primary amino group. Forexample, a fluorescent compound such as Fluoresceinisothiocyanate(FITC), Fluorecein-N-hydroxysuccinimide ester may be reacted with anamino group of an antibiotic, resulting in a Fluorescein-labelledantibiotic. Other antibiotics such as Clindamycin carry a thio-methylgroup which can be coupled to a labelling group. Conditions were foundto mildly substitute the methyl group of Clindamycin with a spacermolecule forming a dithio bridge.

The labelling group may be coupled to the antibiotic via a spacer. Manyspacers are known in the art and may be applied. Using protein chemistrytechniques well known in the art many ways of attaching a spacer andsubsequently attaching a fluorophor are feasible. In a preferredembodiment cysteine is chosen as its primary amino group may readily belabelled with a fluorophor. Molecules with longer carbon backbones andother reactive groups well known in the art may also be chosen aslinker/spacer between any fluorophor and an antibiotic substance.

A list of antibiotics modified with a fluorophor with or without aspacer is compiled in Table 1. It is preferred that the antibiotics (inparticular of Table 1) are labelled with Fluorescein or Atto-495-NSI.

In order to combine the identification with the resistance status, theconditions which allow binding of the labelled antibiotic to its bindingsite in the micro-organism in step (b) may refer to a binding assaywhich is not inhibited by the in-situ hybridisation procedure, enablingeither a concomitant or subsequent determination of both identificationand resistance status in individual cells and cell populations.

A preferred binding site is the PBP2 protein (Penicillin BindingProtein) in Staphylococcus encoded by the mecA gene. In Staphylococcusresistant against beta-lactam antibiotics, the mecA gene encodes amodified PBP2 protein (PBP2′ or PBP2a) with low affinity for methicillinand all β-lactam antibiotics. Thus, in a more preferred embodiment, themicro-organism is a MRSA strain harbour the mecA gene, which encodes amodified PBP2 protein (PBP2′ or PBP2a) with low affinity for methicillinand all β-lactam antibiotics, and the antibiotic is a beta-lactamantibiotic.

A further preferred application is for the determination of resistancedue to point mutations in the 23s ribosomal RNA. The point mutations atdifferent position induce resistance to a wide array of antibiotics suchas Macrolides, Ketolides, Tetracyclin, Thiazolantibiotics, Lincosamin,Chloramphenicol, Streptogramin, Amecitin, Animosycin, Sparsoycin andPuromycin. Detailed effects of respective point mutations are listed inTable 3. Point mutations at different positions of the 23S rRNA cangenerate an iso-phenotype. It would require an array of oligo-nucleotideprobes to cover all possibilities. This invention offers a costeffective and efficient way of detecting antibiotic resistanceirrespective of the position of the mutation.

Another preferred application is the detection of the binding ofVancomycin to surface proteins of Staphylococcus aureus which areanchored to the cell wall peptidoglycan. Vancomycin resistantStaphylococci bind the antibiotic to such an extent that it rendersVancomycin ineffective. Labelled Vancomycin therefore will preferablybind to resistant organisms.

In the present invention, the amount of detectable label in themicro-organism corresponds to the signal of the labelling group of theantibiotic. The amount of detectable label may be directly proportionalto the signal obtained from the labeling group.

The method of the present invention may comprise steps to removelabelling groups which have been cleaved off from the antibiotic or/andto remove labelled antibiotic which is not bound to a micro-organism.Such steps may improve the signal-to-noise ratio.

In step (c) of the method of the present invention, the label may bedetected by any suitable method known in the art. The reading of theassay may require a resolution down to the individual cell. Preferably,the label is detected via epifluorescence microscopy, flow cytometry,laser scanning devices, time resolved fluorometry, luminescencedetection, isotope detection, hyper spectral imaging scanner, SurfacePlasmon Resonance or/and another evanscesence based reading technology.

In step (d) of the method of the present invention, alteration of theamount of detectable label may be an increase of detectable label or adecrease of detectable label. In the method of the present invention,the antibiotic resistance to be detected is predetermined by theprovision of a labelled antibiotic in step (a). Table 4 indicatesresistance mechanisms against commonly known antibiotics in clinicallyrelevant micro-organisms. From the resistance mechanism of a particularpredetermined micro-organism, such as indicated in Table 4, is can bededuced which combination of micro-organism/antibiotic resistance areexpected to show an increased amount of detectable label in antibioticresistant cells, and which combination shows a decreased amount ofdetectable label. For instance, a decrease of the amount of detectablelabel is expected in micro-organisms resistant against β-lactamantibiotics or macrolides, such as MSRA, ORSA, etc. An increased amountof detectable label is expected in VRSA. A decrease of detectable labelis expected in Vancomycin resistant Enterococci, due to the differentresistance mechanism as in VRSA. Further details can be found in Table4.

In the present invention, the predetermined microorganism in itsnon-resistant form can be employed as a reference to determine if theamount of detectable label is altered (decreased or increased). Thepredetermined microorganism in its non-resistant form may be added tothe sample, or may be presented in a separate preparation. Thepredetermined microorganism in its non-resistant form may carry at leastone further label. Any label as described herein may be employed,provided it is suitable for discrimination from the label of theantibiotic or/and other micro-organisms present in the assay of thepresent invention. The amount of detectable label in a predeterminedmicroorganism in its non-resistant form may also be provided in the formof specific values or ranges of the amount of detectable label for oneor more combinations of the micro-organism, an antibiotic and alabelling group, for instance in the form of a data sheet. Inparticular, a kit of the present invention may comprise saidpredetermined micro-organism in its non-resistant form or/and said datasheet.

The method of the present invention may also employ the predeterminedmicro-organism in its resistant form as a further control, or specificvalues or ranges of the amount of detectable label in a predeterminedmicroorganism in its resistant form for one or more combinations of themicro-organism, an antibiotic and a labelling group, for instance in theform of a data sheet. The predetermined microorganism in its resistantform may carry at least one further label. Any label as described hereinmay be employed, provided it is suitable for discrimination from thelabel of the antibiotic or/and other micro-organisms present in theassay of the present invention. In particular, a kit of the presentinvention may comprise said predetermined micro-organism in itsresistant form or/and said data sheet.

The decrease of the amount of detectable label may be at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, or at least 90% with respect tothe amount of detectable label in the predetermined micro-organism inits non-resistant form. In particular, a micro-organism to be identifiedmay be a micro-organism essentially not carrying the label.

The increase of the amount of detectable label may be at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 150%, or at least 200% with respect to the amount of detectablelabel in the predetermined micro-organism in its non-resistant form.

In yet another embodiment of the present invention, the method comprisesidentification of the micro-organism in the biological sample.“Identification” in the context of the present invention refers toidentification of individual microbial cells as belonging to aparticular taxonomic category, such as species, genus, family, classor/and order, etc. Identification can be performed based onmorphological or/and biochemical classifications.

A probe may be employed for identifying the micro-organism. It ispreferred to identify the predetermined micro-organism by a labellednucleic acid, in particular a labelled oligonucleotide, capable ofspecifically hybridising with a nucleic acid in the micro-organism underin-situ conditions. The labelled oligonucleotide may have a length of upto 50 nucleotides. More preferred is identification of themicro-organism by fluorescence in-situ hybridisation (FISH). Thesepreferred and more preferred embodiments allow the detection of theantibiotic phenotype at the molecular level while maintaining in-situhybridisation conditions to allow the simultaneous and unambiguousidentification via in-situ hybridisation and the detection of anantibiotic resistance phenotype within the same cell—even if in a mixedpopulation.

An in-situ hybridisation protocol may be applied as laid down in patentapplication EP 06 021 267.7, which is incorporated herein by reference.The incubation with a labelled antibiotic may be performed attemperatures below the T_(m) of the hybridised probe. In a preferredembodiment the temperature is between about 25 and about 65° C., in amore preferred embodiment the temperature is between about 35° C. andabout 59° C. In an even more preferred embodiment, the temperature is atabout 52° C. The incubation time is preferably between about 1 and about30 minutes. In a more preferred embodiment the incubation is made forabout 15 minutes. After the incubation the slide may be submerged in 50%ethanol followed by a bath in pure ethanol. Both steps may be run forbetween about 1 and about 10 minutes. The preferred length of incubationis between about 2 and about 6 minutes. It is more preferred to incubateabout 4 minutes. The slides may then be air-dried (e.g. on a hot plate)and the cells may be embedded in a balanced salt mounting medium.

The microorganism may be detected by any suitable method known in theart. The reading of the assay may require a resolution down to theindividual cell. In particular, the micro-organism is detected viaepifluorescence microscopy, flow cytometry, laser scanning devices, timeresolved fluorometry, luminescence detection, isotope detection, hyperspectral imaging scanner, Surface Plasmon Resonance or/and anotherevanscesence based reading technology.

Preferably, in-situ hybridisation is combined with detection ofantibiotic resistance. More preferably, FISH is combined with detectionof antibiotic resistance.

It is also preferred that the predetermined micro-organism is identifiedin step (c) of the method of the present invention.

It is preferred that the identification of the micro-organism and thedetection of the labelled antibiotic in the micro-organism are runsubsequently.

In an alternative preferred embodiment, the identification of themicro-organism and the detection of the labelled antibiotic in themicro-organism are run concurrently. In this embodiment, the labelledantibiotic may be added to the hybridisation buffer. After theincubation the further treatment is performed as disclosed herein forthe detection of the micro-organism. Most preferably, in-situhybridisation and FISH, respectively, and detection of the antibioticresistance are performed simultaneously.

Preferably, the same detection method, such as epifluorescencemicroscopy, flow cytometry, laser scanning devices or another methoddescribed herein, may be employed for both the identification of themicro-organism and the detection of the labelled antibiotic in themicro-organism.

In-situ hybridisation and enzyme or receptor assays conventionally callfor specific environments for their respective assays of the state ofthe art. It was therefore surprising that it was possible to

-   1. prepare the cells for in-situ hybridisation with pores of    sufficient size to allow passage of up to 50-mer oligo-nucleotides-   2. make membrane proteins accessible for labelled antibiotics-   3. maintain the integrity of both said proteins and ribosomes to    allow the specific binding of antibiotics labelled with fluorophores-   4. Find sufficient binding sites to generate a signal visible under    an epifluorescence microscope, in particular under uniform    conditions.

It is preferred to use in the method of the present invention anoligo-nucleotide with a fluorophor emitting at a predeterminedwavelengths range together with an antibiotic labelled with anotherfluorophor emitting at a wavelengths range, so that the two fluorophorscan be discriminated by luminescence detection. For instance, one of thefluorphors, such as Fluorescein, may emit a green signal, and the otherfluorphor may emit a red signal. A list of antibiotics modified with afluorophor is compiled in Table I.

In a most preferred embodiment, the method of the present inventioncomprises identification of a Methicillin Resistant Staphylococcusaureus (MRSA) by in-situ hybridisation simultaneously with the detectionof the expression of the Penicillin Binding Protein 2 by binding alabelled β-Lactam antibiotic.

In the case of MRSA it is even possible to differentiate betweennosocomial and community acquired MRSA by analysing the sensitivitytowards Clindamycin or to Trimethoprim sulfa. Community acquiredMethicillin resistant Staphylococcus aureus strains maintain theirsensitivity towards Clindamycin or to Trimethoprim sulfa. Thedifferentiation is then via the respective binding capacity profiles. Inyet another most preferred embodiment, the method of the presentinvention thus comprises identification of a Methicillin ResistantStaphylococcus aureus (MRSA) by in-situ hybridisation simultaneouslywith the differentiation between nosocomial and community acquired MRSAvia the respective ability to bind labelled Clindamycin or Trimethoprimsulfa.

In yet another most preferred embodiment, the method of the presentinvention comprises identification of a Vancomycin ResistantStaphylococcus aureus (VRSA) by in-situ hybridisation simultaneouslywith the ability to bind labelled Vancomycin.

In yet another most preferred embodiment, the method of the presentinvention comprises identification of a Vancomycin ResistantStaphylococcus (VRS) by in-situ hybridisation simultaneously with theability to bind labelled Vancomycin.

In yet another most preferred embodiment, the method of the presentinvention comprises identification of a Vancomycin Resistant Enterococci(VRE) by in-situ hybridisation simultaneously with the ability to bindlabelled Vancomycin.

In yet another most preferred embodiment, the method of the presentinvention comprises detection of a resistance towards β-lactamantibiotics due to the secretion of β-lactamases (ESBL) by the revealingof the presence of said enzyme by the binding of labelled clavulanicacid together with the identification of Gram negative micro-organisms.

Clavulanic acid is a beta-lactamase inhibitor sometimes combined withpenicillin group antibiotics to overcome certain types of antibioticresistance. Specifically, it is used to overcome resistance in bacteriathat secrete beta-lactamase enzymes, which otherwise inactivate mostpenicillins. Most commonly, the potassium salt potassium clavulanate iscombined with amoxicillin. Clavulanic acid is a competitive inhibitor ofbeta-lactam antibiotics. When labelled with a fluorophor it will detectthe of presence beta-lactamases.

In yet another most preferred embodiment, the method of the presentinvention comprises detection of a resistance towards 1′-lactamantibiotics due to the secretion of metalo-β-lactamases (MBL) by therevealing of the presence of said enzyme by the binding of labelledimipenem together with the identification of Gram negativemicro-organisms.

In yet another most preferred embodiment, the method of the presentinvention comprises detection of a resistance to macrolides, lincosamideand streptogramin (MLS) via the binding of labelled erythromycin or/andClindamycin together with the identification of Streptococci.

In yet another most preferred embodiment, the method of the presentinvention comprises identification of a drug resistant Streptococcuspneumoniae (DRSP) with FISH together with the respective resistancetowards beta-lactams and macrolides.

In yet another most preferred embodiment, the method of the presentinvention comprises detection of high level Aminoglycoside resistantEnterococci (HLAR) via FISH and labelled Gentamycin.

The biological sample comprising the predetermined micro-organisms maybe pretreated in order to facilitate binding of the labelled antibioticand optionally identification of the micro-organism.

The biological sample may be heat-fixed on a slide according to theirdesignated probes (labelled antibiotic and optionally a probe fordetecting the micro-organism), for instance at about 45 to about 65° C.,preferably at about 50 to about 55° C., more preferably at about 52° C.

If the micro-organism is a Gram positive bacterium, it may be perforatedby a suitable buffer. Gram positive cells may be perforated with abacteriocin or/and a detergent. In a preferred embodiment a lantibioticis combined with a biological detergent, and a specially preferredembodiment NISIN is combined with Saponin. In addition lytic enzymessuch as Lysozyme and Lysostaphin may be applied. Lytic enzymes may bebalanced into the equation. If the sample is treated with ethanol, theconcentration of the active ingredients may be balanced with respect totheir subsequent treatment in ethanol. In a more preferred embodimentthe concentration of Lysozyme, Lysostaphin, Nisin and Saponin isbalanced to cover all Gram positive organisms with the exception ofMycobacteria.

An example of a most preferred Gram Positive Perforation Buffer is givenin Table 2. It is contemplated that variations of the amounts andconcentrations, and application temperatures and incubation times arewithin the skill in the art.

If the micro-organism is a yeast or a mould, it may be perforated by asuitable buffer. Surprisingly it was found that the cell walls of yeastsand moulds did not form reproducible pores when treated followingprocedures well known in the art. These procedures frequently renderedboth false positive and false negative results. A reliable solution is apreferred buffer comprising a combination of a peptide antibiotic,detergent, complexing agent, and reducing agent. A more preferred buffercomprises the combination of a mono-valent salt generating a specificosmotic pressure, a bacteriocin, a combination of biological andsynthetic detergents, a complexing agent for divalent cations, and anagent capable of reducing disulfide bridges. A further surprisingimprovement was achieved by adding proteolytic enzymes specific forprokaryotes. In an even more preferred buffer, Saponin, SDS, Nisin,EDTA, DTT were combined with Lysozyme and a salt, for in stance in aconcentration of about 150 to about 250 mM, more preferably about 200 toabout 230 mM, most preferably about 215 mM.

An example of a most preferred Yeast Perforation Buffer is given inTable 2. It is contemplated that variations of the amounts andconcentrations, and application temperatures and incubation are withinthe skill in the art.

In yet another preferred embodiment, the method of the present inventionis a diagnostic method.

Yet another aspect of the present invention is a kit suitable fordetecting an antibiotic resistance in a predetermined micro-organism,comprising

-   (a) a labelled antibiotic, and-   (b) optionally a probe suitable for identification of the    micro-organism in the biological sample.

The kit of the present invention is suitable in the method of thepresent invention. The labelled antibiotic may be a labelled antibioticas described herein in the context of the method of the presentinvention.

The probe may be any probe suitable for identification of amicroorganism. It is preferred that the probe is a labelled nucleicacid, in particular a labelled oligonucleotide, capable of specificallyhybridising with a nucleic acid in the micro-organism under in-situconditions. The oligonucleotide may have a length up to 50 nucleotides.

As indicated above, the kit may comprise further components, such as adata sheet providing information about the amount of detectable label inat least one combination of micro-organism, antibiotic and label, or asample of a predetermined micro-organism in its non-resistant or/andresistant form, e.g. for control purposes.

Yet another aspect of the present invention is the use of a labelledantibiotic for the detection of an antibiotic resistance in apredetermined micro-organism in a biological sample.

The present invention is further illustrated by the following examplesand the following tables.

Table 1 describes the antibiotics and examples of labelling suitable inthe method of the present invention.

Table 2 describes the composition of perforation buffers employed in thepresent invention.

Table 3: Antibiotic resistance due to mutations on the 23S rRNA.

Table 4: Antibiotic resistance mechanism in micro-organisms andalteration in the amount of labelled antibiotics in resistantmicro-organisms.

EXAMPLE 1

The antibiotics of Table I were labelled with FITC and purified as iswell known in the art. Clindamycin was modified by substituting themethyl group attached to the X′—S with cysteine via an S—S bond. Theattached cysteine was then labelled with Fluorescamin either via anN-hydroxy-succinimide ester or FITC and purified with methods well knownin the art.

EXAMPLE 2

An antibiotic resistance, such as a resistance against penicillin, maybe detected in a protocol comprising the steps:

1 Apply the biological sample to slide, e.g. 10 μl2 Dry, for instance at 52° C.3 Add perforation buffer, e.g. 10 μl

4 Dry

5 Add reconstituted probe mix (e.g. 9 μl)6 Add antibiotic (e.g. FITC-penicillin)

7 Incubate, e.g. for 15 min at 52° C. 8 EtOH/Stop mix (e.g. 50%:50%),e.g. for 5 min at RT

9 Ethanol, e.g. 99% ethanol for 5 min

10 Dry

11 Balanced Salt Mounting Medium (e.g. one small drop)

12 Read EXAMPLE 3

Table 4 indicates the alteration of the amount of detectable labelledantibiotics in clinically relevant micro-organisms in its resistant formrelative to its non-resistant form. The amount is expressed in % changeof fluorescence (decrease and increase, respectively) of an antibioticwhich carries a fluorescent label.

TABLE 1 Antibiotics Generic Name Examples of Labelling agentAminoglycosides Amikacin FITC Atto-495-NSI Gentamicin FITC Atto-495-NSIKanamycin FITC Atto-495-NSI Neomycin FITC Atto-495-NSI Netilmicin FITCAtto-495-NSI Streptomycin FITC Atto-495-NSI Tobramycin FITC Atto-495-NSIFITC Atto-495-NSI Carbacephem Loracarbef FITC Atto-495-NSI CarbapenemsErtapenem FITC Atto-495-NSI Imipenem FITC Atto-495-NSI Cilastatin FITCAtto-495-NSI Meropenem FITC Atto-495-NSI Cephalosporins First generationCefadroxil FITC Atto-495-NSI Cefazolin FITC Atto-495-NSI Cephalexin FITCAtto-495-NSI Cephalosporins Second generation Cefaclor FITC Atto-495-NSICefamandole FITC Atto-495-NSI Cefoxitin FITC Atto-495-NSI Cefprozil FITCAtto-495-NSI Cefuroxime FITC Atto-495-NSI Cephalosporins Thirdgeneration Cefixime FITC Atto-495-NSI Cefdinir FITC Atto-495-NSICefditoren FITC Atto-495-NSI Cefoperazone FITC Atto-495-NSI CefotaximeFITC Atto-495-NSI Cefpodoxime FITC Atto-495-NSI Ceftazidime FITCAtto-495-NSI Ceftibuten FITC Atto-495-NSI Ceftizoxime FITC Atto-495-NSICeftriaxone FITC Atto-495-NSI Cefsulodine FITC Atto-495-NSICephalosporins Fourth generation Cefepime FITC Atto-495-NSIGlycopeptides Teicoplanin FITC Atto-495-NSI Vancomycin FITC Atto-495-NSIMacrolides Azithromycin Coupling of Coupling of Clarithromycin FITC withErythro Dirithromvcin Erythro mycylamine with Erythromycin mycylamineAtto-495-NSI Roxithromycin Troleandomycin Monobactam Aztreonam FITCAtto-495-NSI Penicillins Amoxicillin FITC Atto-495-NSI Ampicillin FITCAtto-495-NSI Azlocillin Carbenicillin Cloxacillin DicloxacillinFlucloxacillin Mezlocillin Nafcillin Penicillin Piperacillin TicarcillinPolypeptides Bacitracin FITC Atto-495-NSI Colistin FITC Atto-495-NSIPolymyxin B FITC Atto-495-NSI Quinolones Ciprofloxacin EnoxacinGatifloxacin Levofloxacin Lomefloxacin Moxifloxacin NorfloxacinOfloxacin Trovafloxacin Sulfonamides Mafenide FITC Atto-495-NSIProntosil FITC Atto-495-NSI (archaic) Sulfacetamide FITC Atto-495-NSISulfamethizole FITC Atto-495-NSI Sulfanilimide FITC Atto-495-NSI(archaic) Sulfasalazine FITC Atto-495-NSI Sulfisoxazole FITCAtto-495-NSI Trimethoprim sulfa FITC Atto-495-NSI Trimethoprim FITCAtto-495-NSI Sulfamethoxazole FITC Atto-495-NSI Co-trimoxazole FITCAtto-495-NSI TMP-SMX FITC Atto-495-NSI Tetracyclines Demeclocycline FITCAtto-495-NSI Doxvcycline FITC Atto-495-NSI Minocycline FITC Atto-495-NSIOxytetracvcline FITC Atto-495-NSI Tetracycline FITC Atto-495-NSI OthersChloramphenicol Clindamycin Cysteine + FITC Cystein + Atto-495-NSIEthambutol Fosfomycin Furazolidone Isoniazid Linezolid MetronidazoleMupirocin Nitrofurantoin Platensimvcin Pyrazinamide Quinupristin/Dalfopristin Rifampin Spectinomycin Amphotericin B FITC Atto-495-NSIFlucanazole Fluoropyrimidins

TABLE 2 Gram-Positive Perforation Buffer- 50 μg/ml Saponin 5 μg/ml Nisin20 mM Tris pH 8 100 μg/m Lysozym 50 μg/ml Lysostaphin H ₂ O Workingsoln. Yeast Perforation Buffer 500 μg/ml Saponin 10 μg/ml Nisin 50 mMTris pH 8.3 215 mM NaCl 0.1 % SDS 5 mM EDTA 10 mM DTT 100 μg/ml LysozymeH ₂ O

TABLE 3 Compilation of antibiotic resistance due to mutations on the 23SrRNA Type of Posi- RNA tion Alteration(s) Phenotype Organism Reference23S 2032 AG to GA Clr/Azm/Ery-R Helicobacter Húlten, K., A. Gibreel, O.Sköld, and L. Engstrand. pylori 1997. Macrolide resistance inHelicobacter pylori: mechanism and stability in strains fromclarithromycin-treated patients. Antimicrob. Agents Chemother. 41:2550-2553. 23S 2058 A to C Clr-R Helicobacter Stone, G. G., D.Shortridge, R. K. Flamm, J. Versalovic, pylori J. Beyer, K. Idler, L.Zulawinski, and S. K. Tanaka. 1996. Identification of a 23S rRNA genemutation in clarithromycin-resistant Helicobacter pylori. Helicobacter.1: 227-228. 23S 2058 A to C Mac-R, Lin-R Helicobacter Occhialini, A., M.Urdaci, F. Doucet-Populaire, pylori C. M. Bébéar, H. Lamouliatte, and F.Mégraud. 1997. Macrolide resistance in Helicobacter pylori: rapiddetection of point mutations and assays of macrolide binding toribosomes. Antimicrob. Agents Chemothe 23S 2058 A to C MLSB-RHelicobacter Wang, G., and D. E. Taylor. 1998. Site-specific pylorimutations in the 23S rRNA gene of Helicobacter pylori confer two typesof resistance to macrolide- lincosamide-streptogramin B antibiotics.Antimicrob. Agents Chemother. 42: 1952-1958. 23S 2058 A to C Cla-RHelicobacter Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,pylori C. M. Vandenbroucke-Grauls, and J. G. Kusters. 1998. Explainingthe bias in the 23S rRNA gene mutations associated with clarithromycinresistance in clinical isolates of Helicobacter pylori. Antimi 23S 2058A to G Cla-R Helicobacter Versalovic, J., D. Shortridge, K. Kibler, M.V. Griffy, pylori J. Beyer, R. K. Flamm, S. K. Tanaka. D. Y. Graham, andM. F. Go. 1996. Mutations in 23S rRNA are associated with clarithromycinresistance in Helicobacter pylori. Antimicrob. Agents Chemother. 40:423S 2058 A to G Mac-R, Lin-R Helicobacter Occhialini, A., M. Urdaci, F.Doucet-Populaire, pylori C. M. Bébéar, H. Lamouliatte, and F. Mégraud.1997. Macrolide resistance in Helicobacter pylori: rapid detection ofpoint mutations and assays of macrolide binding to ribosomes.Antimicrob. Agents Chemothe 23S 2058 A to G MLSB-R Helicobacter Wang,G., and D. E. Taylor. 1998. Site-specific pylori mutations in the 23SrRNA gene of Helicobacter pylori confer two types of resistance tomacrolide- lincosamide-streptogramin B antibiotics. Antimicrob. AgentsChemother. 42: 1952-1958. 23S 2058 A to G Cla-R HelicobacterDebets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers, pylori C. M.Vandenbroucke-Grauls, and J. G. Kusters. 1998. Explaining the bias inthe 23S rRNA gene mutations associated with clarithromycin resistance inclinical isolates of Helicobacter pylori. Antimi 23S 2058 A to U MLSB-RHelicobacter Wang, G., and D. E. Taylor. 1998. Site-specific pylorimutations in the 23S rRNA gene of Helicobacter pylori confer two typesof resistance to macrolide- lincosamide-streptogramin B antibiotics.Antimicrob. Agents Chemother. 42: 1952-1958. 23S 2058 A to U Cla-RHelicobacter Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,pylori C. M. Vandenbroucke-Grauls, and J. G. Kusters. 1998. Explainingthe bias in the 23S rRNA gene mutations associated with clarithromycinresistance in clinical isolates of Helicobacter pylori. Antimi 23S 2059A to C Mac-R, Lin-R, Helicobacter Wang, G., and D. E. Taylor. 1998.Site-specific SB-S pylori mutations in the 23S rRNA gene of Helicobacterpylori confer two types of resistance to macrolide-lincosamide-streptogramin B antibiotics. Antimicrob. Agents Chemother.42: 1952-1958. 23S 2059 A to C Clr-R Helicobacter Debets-Ossenkopp, Y.J., A. B. Brinkman, E. J. Kuipers, pylori C. M. Vandenbroucke-Grauls,and J. G. Kusters. 1998. Explaining the bias in the 23S rRNA genemutations associated with clarithromycin resistance in clinical isolatesof Helicobacter pylori. Antimi 23S 2059 A to G Clr-R HelicobacterVersalovic, J., D. Shortridge, K. Kibler, M. V. Griffy, pylori J. Beyer,R. K. Flamm, S. K. Tanaka. D. Y. Graham, and M. F. Go. 1996. Mutationsin 23S rRNA are associated with clarithromycin resistance inHelicobacter pylori. Antimicrob. Agents Chemother. 40:4 23S 2059 A to GMac-R, Lin-R Helicobacter Occhialini, A., M. Urdaci, F.Doucet-Populaire, pylori C. M. Bébéar, H. Lamouliatte, and F. Mégraud.1997. Macrolide resistance in Helicobacter pylori: rapid detection ofpoint mutations and assays of macrolide binding to ribosomes.Antimicrob. Agents Chemothe 23S 2059 A to G Mac-R, Lin-R, HelicobacterWang, G., and D. E. Taylor. 1998. Site-specific SB-S pylori mutations inthe 23S rRNA gene of Helicobacter pylori confer two types of resistanceto macrolide- lincosamide-streptogramin B antibiotics. Antimicrob.Agents Chemother. 42: 1952-1958. 23S 2059 A to G Cla-R HelicobacterDebets-Ossenkopp. Y. J., A. B. Brinkman E. J. Kuipers, pylori C. M.Vandenbroucke-Grauls, and J. G. Kusters. 1998. Explaining the bias inthe 23S rRNA gene mutations associated with clarithromycin resistance inclinical isolates of Helicobacter pylori. Antimi 23S 754 “U to A”Resistant to low E. coli Xiong L, Shah S, Mauvais P, Mankin AS.concentrations of ketolide 1999. A ketolide resistance mutation inHMR3647; resistant to domain II of 23S rRNA reveals the erythromycin b.proximity of hairpin 35 to the peptidyl transferase center. MolecularMicrobiology 31 (2): 633-639. 23S 754 “U to A” Confers macrolide and E.coli Hansen L. H., Mauvais P, Douthwaite S. ketolide resistance. 1999.The macrolide-ketolide antibiotic binding site is formed by structuresin domain II and V of 23S ribosomal RNA. Molecular Microbiology 31 (2):623-631. 23S 754 U to A Ery-LR, Tel-LR Escherichia Xiong, L., S. Shah,P. Mauvais, and A. S. Mankin. coli 1999. A ketolide resistance mutationin domain II of 23S rRNA reveals the proximity of hairpin 35 to thepeptidyl transferase centre. Mol. Microbiol. 31: 633-639. 23S 1005 C toG Slow growth under natural E. coli 1) Rosendahl, G. and Douthwaite, S.promoter; (with 2058G and (1995) Nucleic Acids Res. 23, 2396-2403.erythromycin) severe growth 2) Rosendahl, G., Hansen, L. H., andretardation. A Douthwaite, S. (1995) J. Mol. Biol. 249, 59-68. 23S 1005C to G Slow growth under pL E. coli 1) Rosendahl, G. and Douthwaite, S.promoter; (with 2058G and (1995) Nucleic Acids Res. 23, 2396-2403.erythromycin) Erys. a Double 2) Rosendahl, G., Hansen, L. H., and mutant(C1005G/C1006U) Douthwaite, S. (1995) J. Mol. Biol. 249, 59-68. 23S 1006C to U Slow growth under pL E. coli 1) Rosendahl, G. and Douthwaite, S.promoter; (with 2058G and (1995) Nucleic Acids Res. 23, 2396-2403.erythromycin) Erys. a Double 2) Rosendahl, G., Hansen, L. H., and mutant(C1005G/C1006U) Douthwaite, S. (1995) J. Mol. Biol. 249, 59-68. 23S 1006C to U Lethal under natural E. coli 1) Rosendahl, G. and Douthwaite, S.promoter; under pL (1995) Nucleic Acids Res. 23, 2396-2403. promoter;(with 2058G and 2) Rosendahl, G., Hansen, L. H., and erythormycin) Erys.A Douthwaite, S. (1995) J. Mol. Biol. 249, 59-68. 23S 1056 G to ABinding of both L11 and E. coli Ryan, P. C. and Draper, D. E. (1991)Proc. thiostrepton is weakened in Natl. Acad. Sci. USA 88, 6308-6312.RNA fragment. B 23S 1056 G to A Stoichiometric L11 binding.b E. coli 1)Douthwaite, S. and Aagaard, C. (1993) (with 2058G and J. Mol. Biol. 232,725-731. 2) Rosendahl, G. erythromycin) Reduced and Douthwaite, S.(1995) Nucleic growth rate. a Acids Res. 23, 2396-2403. 23S 1056 G to CBinding of thiostrepton is E. coli Ryan, P. C. and Draper, D. E. (1991)Proc. weakened in RNA Natl. Acad. Sci. USA 88, 6308-6312. fragments. B23S 1064 C to U Stoichiometric L11 binding. b E. coli 1) Douthwaite, S.and Aagaard, C. (1993) (with 2058G and J. Mol. Biol. 232, 725-731. 2)Rosendahl, G. erythromycin) Reduced and Douthwaite, S. (1995) Nucleicgrowth rate. a Acids Res. 23, 2396-2403. 23S 1067 A to U Normal growthE. coli 1) Spahn, C., Remme, J., Schafer, M. and Nierhaus, K. (1996). J.Biol. Chem. 271: 32849-32856. 2) Spahn, C., Remme, J., Schafer, M. andNierhaus, K. (1996). J. Biol. Chem. 271: 32857-32862. 23S 1067 A to GThiostrepton resistance in Halobacterium Hummel, H., and A. Böck. (1987)Halobacterium sp. Biochimie 69: 857-861. 23S 1067 A to U Thiostreptonresistance in Halobacterium Hummel, H., and A. Böck. (1987)Halobacterium sp. Biochimie 69: 857-861. 23S 1067 A to U A to C or Uconfers high E. coli 1) Thompson, J. and Cundliffe, E. (1991) levelresistance to Biochimie 73: 1131-1135. 2) Thompson, J., thiostrepton,whereas A to G Cundliffe, E. and Dahlberg, A. E. (1988) confersintermediate level J. Mol. Biol. 203: 457-465. 3) Lewicki, B. T. U.,resistance; drug binding Margus, T., Remme, J. and affinity is reducedsimilarly. Nierhaus, K. H. (1993) J. Mol. Biol. 231, a, b Expression byhost RNA 581-593. 4) LAST polymerase results in formation of activeribosomal subunits in vivo. A 23S 1067 A to C A to C or U confers highE. coli 1) Thompson, J. and Cundliffe, E. (1991) level resistance toBiochimie 73: 1131-1135. 2) Thompson, J., thiostrepton, whereas A to GCundliffe, E. and Dahlberg, A. E. (1988) confers intermediate level J.Mol. Biol. 203: 457-465. 3) Lewicki, B. T. U., resistance; drug bindingMargus, T., Remme, J. and affinity is reduced similarly. Nierhaus, K. H.(1993) J. Mol. Biol. 231, a, b Expression by host RNA 581-593. 4) LASTpolymerase results in formation of active ribosomal subunits in vivo. A23S 1067 A to G A to C or U confers high E. coli 1) Thompson, J. andCundliffe, E. (1991) level resistance to Biochimie 73: 1131-1135. 2)Thompson, J., thiostrepton, whereas A to G Cundliffe, E. and Dahlberg,A. E. (1988) confers intermediate level J. Mol. Biol. 203: 457-465. 3)Lewicki, B. T. U., resistance; drug binding Margus, T., Remme, J. andaffinity is reduced similarly. Nierhaus, K. H. (1993) J. Mol. Biol. 231,a, b Expression by host RNA 581-593. 4) LAST polymerase results information of active ribosomal subunits in vivo. A 23S 1067 “A to U”Constituted 30% of the total E. coli Liiv A, Remme J. 1998. Base-pairingof 23S rRNA pool in the 23S rRNA ends is essential for ribosomalribosomes; exhibited 30% large subunit assembly. J. Mol. Biol. 285:thiostrepton resistance in 965-975. poly (U) translation b. 23S 1068 Gto A Reduced L11 binding. b (with E. coli 1) Rosendahl, G. andDouthwaite, S. 2058G) Lethal when (1995) Nucleic Acids Res. 23,2396-2403. expressed from rrnB or pL 2) Douthwaite, S. and Aagaard, C.promotor in presence of (1993) J. Mol. Biol. 232, 725-731. erythromycin.A 23S 1068 G to A Suppression of 1068A; E. coli Rosendahl, G. andDouthwaite, S. lethality only in absence of (1995) Nucleic Acids Res.23, 2396-2403. erythromycin. a Double mutant (G1068A/G1099A) 23S 1072 Cto U Lethal when expressed from E. coli Rosendahl, G. and Douthwaite, S.rrnB or pL promotor in (1995) Nucleic Acids Res. 23, 2396-2403. presenceof erythromycin. a 23S 1137 G to A With 2058G and erythromycin, E. coliRosendahl, G., Hansen, L. H., and lethal when expressed from rrnBDouthwaite, S. (1995) J. Mol. Biol. promoter. 249, 59-68. 23S 1137 G toA Restores normal growth under pL E. coli Rosendahl, G., Hansen, L. H.,and promotor; (With 2058G and Douthwaite, S. (1995) J. Mol. Biol.erythromycin) Eryr. Double 249, 59-68. mutant (G1137A/C1006U) 23S 1137 Gto A With 2058G and erythromycin, E. coli Rosendahl, G., Hansen, L. H.,and lethal when expressed from rrnB Douthwaite, S. (1995) J. Mol. Biol.249, promoter. Double mutant 59-68. (G1137A/G1138C) 23S 1138 G to C With2058G and erythromycin, E. coli Rosendahl, G., Hansen, L. H., and lethalwhen expressed from rrnB Douthwaite, S. (1995) J. Mol. Biol. 249,promoter. 59-68. 23S 1138 G to C With 2058G and erythromycin, E. coliRosendahl, G., Hansen, L. H., and lethal when expressed from rrnBDouthwaite, S. (1995) J. Mol. Biol. 249, promoter. Double mutant 59-68.(G1138C/G1137A) 23S 1207 C to U Erythromycin resistant. a Double E. coliDam, M., Douthwaite, S., Tenson, T. mutant (C1207U/C1243U) and Mankin,A. S. (1996) J. Mol. Biol. 259, 1-6. 23S 1208 C to U Erythromycinresistant. a Double E. coli Dam, M., Douthwaite, S., Tenson, T. mutant(C1208U/C1243U) and Mankin, A. S. (1996) J. Mol. Biol. 259, 1-6. 23S1211 C to U Erythromycin sensitive. a Double E. coli Dam, M.,Douthwaite, S., Tenson, T. mutant (C1211U/C1208U) and Mankin, A. S.(1996) J. Mol. Biol. 259, 1-6. 23S 1220 G to A Erythromycin resistant. aDouble E. coli Dam, M., Douthwaite, S., Tenson, T. mutant(G1220A/G1239A) and Mankin, A. S. (1996) J. Mol. Biol. 259, 1-6. 23S1221 C to U Erythromycin resistant. a Double E. coli Dam, M.,Douthwaite, S., Tenson, T. mutant (C1221U/C1229U) and Mankin, A. S.(1996) J. Mol. Biol. 259, 1-6. 23S 1221 C to U Erythromycin resistant. aDouble E. coli Dam, M., Douthwaite, S., Tenson, T. mutant(C1221U/C1233U) and Mankin, A. S. (1996) J. Mol. Biol. 259, 1-6. 23S1230 1230 Erythromycin sensitive. a Double E. coli Douthwaite, S.,Powers, T., Lee, J. Y., deletion (1230/1231) and Noller, H. F. (1989) J.Mol. Biol. 209, 655-665. 23S 1231 1231 Erythromycin sensitive. a DoubleE. coli Douthwaite, S., Powers, T., Lee, J. Y., deletion (1231/1230) andNoller, H. F. (1989) J. Mol. Biol. 209, 655-665. 23S 1232 G to AErythromycin sensitive. a Double E. coli Dam, M., Douthwaite, S.,Tenson, T. mutant (G1232A/G1238A) and Mankin, A. S. (1996) J. Mol. Biol.259, 1-6. 23S 1233 C to U Erythromycin sensitive. a E. coli Dam, M.,Douthwaite, S., Tenson, T. and Mankin, A. S. (1996) J. Mol. Biol. 259,1-6. 23S 1234 “del1234/del1235” Erythromycin sensitive. a Double E. coliDouthwaite, S., Powers, T., Lee, J. Y., mutant (U1234C/del1235) andNoller, H. F. (1989) J. Mol. Biol. 209, 655-665. 23S 1234 U to CErythromycin sensitive. a E. coli Douthwaite, S., Powers, T., Lee, J.Y., and Noller, H. F. (1989) J. Mol. Biol. 209, 655-665. 23S 1243 C to UErythromycin resistant. a Double E. coli Douthwaite, S., Powers, T.,Lee, J. Y., mutant (C1243U/C1208U) and Noller, H. F. (1989) J. Mol.Biol. 209, 655-665. 23S 1243 C to U Erythromycin resistant. a Double E.coli Douthwaite, S., Powers, T., Lee, J. Y., mutant (C1243U/C1221U) andNoller, H. F. (1989) J. Mol. Biol. 209, 655-665. 23S 1243 “C to U”Erythromycin resistant. a Double E. coli Douthwaite, S., Powers, T.,Lee, J. Y., mutant (C1243U/C1207). and Noller, H. F. (1989) J. Mol.Biol. 209, 655-665. 23S 1262 A to G With erythromycin; lethal E. coliAagaard, C., and Douthwaite, S. (1994) Proc. Natl. Acad. Sci. USA 91,2989-2993. 23S 1262 A to C With erythromycin; lethal E. coli Aagaard,C., and Douthwaite, S. (1994) Proc. Natl. Acad. Sci. USA 91, 2989-2993.23S 1262 A to U With erythromycin; reduced E. coli Aagaard, C., andDouthwaite, S. (1994) growth rate Proc. Natl. Acad. Sci. USA 91,2989-2993. 23S 1262 A to C With erythromycin; reduced E. coli Aagaard,C., and Douthwaite, S. (1994) growth rate Double mutant Proc. Natl.Acad. Sci. USA 91, 2989-2993. (A1262C/U2017G) 23S 1262 A to GSuppression of growth effects; E. coli Aagaard, C., and Douthwaite, S.(1994) Wild-type growth on Proc. Natl. Acad. Sci. USA 91, 2989-2993.erythromycin Double mutant (A1262G/U2017C) 23S 1262 A to U Suppressionof growth effects; E. coli Aagaard, C., and Douthwaite, S. (1994)Wild-type growth on Proc. Natl. Acad. Sci. USA 91, 2989-2993.erythromycin Double mutant (A1262U/U2017A) 23S 1262 A to U Witherythromycin; reduced E. coli Aagaard, C., and Douthwaite, S. (1994)growth rate Double mutant Proc. Natl. Acad. Sci. USA 91, 2989-2993.(A1262U/U2017G) 23S 1423 G to A Suppressed requirement for E. coliO'Connor, M., Brunelli, C. A., Firpo, M. A., 4.5S RNA in translation ofGregory, S. T., Lieberman, K. R., natural mRNAs by cell extracts. cLodmell, J. S., Moine, H., Van Ryk, D. I. and Dahlberg, A. E. (1995)Biochem. Cell Biology 73, 859-868. 23S 1698 A to G Suppresses 2555mutations E. coli O'Connor & Dahlberg, unpublished 23S 2017 “U to G”Reduced growth E. coli Aaagard, C. and Douthwaite, S. (1994) Proc. rateon Natl. Acad. Sci. USA 91, 2989-2993. eyrthomycin. 23S 2017 “U to C”Reduced growth E. coli Aaagard, C. and Douthwaite, S. (1994) Proc. rateof Natl. Acad. Sci. USA 91, 2989-2993. erythromycin. 23S 2017 “U to A”Reduced growth E. coli Aaagard, C. and Douthwaite, S. (1994) Proc. rateof Natl. Acad. Sci. USA 91, 2989-2993. erythromycin. 23S 2017 “U to C”Reduced growth E. coli Aaagard, C. and Douthwaite, S. (1994) Proc. rateon Natl. Acad. Sci. USA 91, 2989-2993. erythromycin. Double mutation(U2017C/A1262G) 23S 2017 “U to G” Reduced growth E. coli Aaagard, C. andDouthwaite, S. (1994) Proc. rate on Natl. Acad. Sci. USA 91, 2989-2993.erythromycin. Double mutation (U2017G/A1262C) 23S 2017 “U to G” Reducedgrowth E. coli Aaagard, C. and Douthwaite, S. (1994) Proc. rate on Natl.Acad. Sci. USA 91, 2989-2993. erythromycin. Double mutation(U2017G/A1262U) 23S 2032 “G to A” Lincomycin Tobbaco Cseplö, A., Etzold,T., Schell, J., and Schreier, P. H. resistance. chloroplasts (1988) Mol.Genet. 214, 295-299. 23S 2032 “G to A” EryS, Cds, Cms. E. coli 1.Douthwaite, S. (1992) J. Bacteriol. 174, Double mutation 1333-1338. 2.Aaagard, C. and Douthwaite, S. (G2032A/A2058G) (1994) Proc. Natl. Acad.Sci. USA 91, 2989-2993. 3. Vester, B., Hansen, L. H., and Douthwaite, S.(1995) RNA 1, 501-509. 23S 2032 “G to A” Eryhs, Cds, Cms. E. coli 1.Douthwaite, S. (1992) J. Bacteriol. 174, Double mutation 1333-1338. 2.Aaagard, C. and Douthwaite, S. (G2032A/A2058U) (1994) Proc. Natl. Acad.Sci. USA 91, 2989-2993. 3. Vester, B., Hansen, L. H., and Douthwaite, S.(1995) RNA 1, 501-509. 23S 2032 “G to A” Eryr, Cdr, Cmr. E. coli 1.Douthwaite, S. (1992) J. Bacteriol. 174, Double mutation 1333-1338. 2.Aaagard, C. and Douthwaite, S. (G2032A/G2057A) (1994) Proc. Natl. Acad.Sci. USA 91, 2989-2993. 3. Vester, B., Hansen, L. H., and Douthwaite, S.(1995) RNA 1, 501-509. 23S 2032 AG to GA Clr/Azm/Ery-R HelicobacterHúlten, K., A. Gibreel, O. Sköld, and L. Engstrand. pylori 1997.Macrolide resistance in Helicobacter pylori: mechanism and stability instrains from clarithromycin-treated patients. Antimicrob. AgentsChemother. 41: 2550-2553. 23S 2051 “del A” Prevents ErmE E. coli VesterB, Nielsen AK, Hansen LH, Douthwaite S. methylation. c 1998. ErmEMethyltransferase Recognition Elements in RNA Substrates. J. Mol. Biol.282: 255-264. 23S 2052 “A to C” Prevents ErmE E. coli Vester B, NielsenAK, Hansen LH, Douthwaite S. methylation. c 1998. ErmE MethyltransferaseRecognition Elements in RNA Substrates. J. Mol. Biol. 282: 255-264. 23S2052 “A to G” Like A2052C. c E. coli Vester B, Nielsen AK, Hansen LH,Douthwaite S. 1998. ErmE Methyltransferase Recognition Elements in RNASubstrates. J. Mol. Biol. 282: 255-264. 23S 2052 “A to U” Like A2052C. cE. coli Vester B, Nielsen AK, Hansen LH, Douthwaite S. 1998. ErmEMethyltransferase Recognition Elements in RNA Substrates. J. Mol. Biol.282: 255-264. 23S 2057 “G to A” Eryr, Clinidamycin E. coli 1. Ettayebi,M., Prasad, S. M., and Morgan, E. A. (Cd)s, (1985) J. Bacteriol. 162,551-557 2. Chloramphemicol Aaagard, C. and Douthwaite, S. (1994) Proc.(Cm)r; reduces Natl. Acad. Sci. USA 91, 2989-2993. 3. methylation of 23SVester, B., Hansen, L. H., and Douthwaite, S. rRNA by ErmE. (1995) RNA1, 501-509. 23S 2057 “G to A” Eyrr. Chlamydomonas Harris, E. H.,Burkhart, B. D., Gilham, N. W., reinhardtii and Boynton, J. E. (1989)Genetics 123, 281-292. 23S 2057 “G to A” Slightly Eryr; E. coli Vester,B., Hansen, L. H., and Douthwaite, S. reduced (1995) RNA 1, 501-509.methylation. Double mutation (G2057A/C2661U) 23S 2057 “G to A” Eryr,Cdr, Cmr. E. coli 1. Douthwaite, S. (1992) J. Bacteriol. 174, Doublemutation 1333-1338. 2. Aaagard, C. and Douthwaite, S. (G2057A/G2032A)(1994) Proc. Natl. Acad. Sci. USA 91, 2989-2993. 3. Vester B., Hansen,L. H., and Douthwaite, S. (1995) RNA 1, 501-509. 23S 2057 G to A Ery-R,Lin-S Chlamydomonas Harris, E. H., B. D. Burkhart, N. W. Gillham,reinhardtii chl. and J. E. Boynton. 1989. Antibiotic resistancemutations in the chloroplast 16S and 23S rRNA genes of Chlamydomonasreinhardtii: correlation of genetic and physical maps of the chloroplastgenome. Genetics. 23S 2057 G to A Ery-R, M16-S, Lin- Escherichia coliEttayebi, M., S. M. Prasad, and E. A. Morgan. S, SB-S 1985.Chloramphenicol- erythromycin resistance mutations in a 23S rRNA gene ofEscherichia coli. J. Bacteriol. 162: 551-557. 23S 2057 G to A Ery-LR,M16-S Propionibacteria Ross, J. I., E. A. Eady, J. H. Cove, C. E. Jones,A. H. Ratyal, Y. W. Miller, S. Vyakrnam, and W. J. Cunliffe. 1997.Clinical resistance to erythromycin and clindamycin in cutaneouspropionibacteria isolated from acne patients is associated with mutatio23S 2057 GG to AA Ery-R, Lin-R Escherichia coli Douthwaite, S. 1992.Functional interactions within 23S rRNA involving thepeptidyltransferase center. J. Bacteriol. 174: 1333-1338. 23S 2058 “A toG” Eryr, Lincomycin Chlamydomonas Harris, E. H., Burkhart, B. D.,Gilham, N. W., and clindamycin reinhardtii and Boynton, J. E. (1989)Genetics 123, resistance. 281-292. 23S 2058 “A to G” ClarithromycinHelecobacter Versalovic, J., Shortridge, D., Kibler, K., resistancepylori Griffy, M. V., Beyer, J., Flamm, R. K., Tenaka, S. K., Graham, D.Y., and Go, M. F. (1996) Antimicrob. Agents Chemother. 40, 477-480. 23S2058 “A to G” Eryr, Cdr, Cms; E. coli 1. Vester, B. and Garrett, R. A.(1988) abolishes EMBO J. 7, 3577-3587. 2. Aaagard, C. and methylation of23S Douthwaite, S. (1994) Proc. Natl. Acad. Sci. rRNA by ErmE. USA 91,2989-2993. 3. Vester, B., Hansen, L. H., and Douthwaite, S. (1995) RNA1, 501-509. 23S 2058 “A to G” Erythromycin Yeast Sor, F. and Fukuhara,H. (1982) Nucleic resistance. mitochondria Acids Res. 10, 6571-6577. 23S2058 “A to G” Lincomycin Solanum nigrum Kavanagh, T. A., O'Driscoll, K.M., McCabe, P. F., resistance. and Dix, P. J. (1994) Mol. Gen. Genet.242, 675-680. 23S 2058 “A to G” Lincomycin Tobacco Cseplö, A., Etzold,T., Schell, J., and resistance. chloroplasts Schreier, P. H. (1988) Mol.Genet. 214, 295-299. 23S 2058 “A to G” EryS, Cds, Cms. E. coli 1.Douthwaite, S. (1992) J. Bacteriol. 174, Double mutation 1333-1338. 2.Aaagard, C. and Douthwaite, S. (A2058G/G2032A) (1994) Proc. Natl. Acad.Sci. USA 91, 2989-2993. 3. Vester, B., Hansen, L. H., and Douthwaite, S.(1995) RNA 1, 501-509. 23S 2058 “A to U” Eryhs, Cds, Cms. E. coli 1.Douthwaite, S. (1992) J. Bacteriol. 174, Double mutation 1333-1338. 2.Aaagard, C. and Douthwaite, S. (A2058U/G2032A) (1994) Proc. Natl. Acad.Sci. USA 91, 2989-2993. 3. Vester, B., Hansen, L. H., and Douthwaite, S.(1995) RNA 1, 501-509. 23S 2058 “A to C” Confers E. coli Hansen LH,Mauvais P, Douthwaite S. resistance to the 1999. The macrolide-kelotideantibiotic MLS drugs and binding site is formed by structures inchloramphenicol. domains II and V of 23S ribosomal RNA. MolecularMicrobiology 31 (2): 623-631. 23S 2058 “A to G” Like A2058C E. coliHansen LH, Mauvais P, Douthwaite S. 1999. The macrolide-kelotideantibiotic binding site is formed by structures in domains II and V of23S ribosomal RNA. Molecular Microbiology 31 (2): 623-631. 23S 2058 “Ato U” Like A2058C E. coli Hansen LH, Mauvais P, Douthwaite S. 1999. Themacrolide-kelotide antibiotic binding site is formed by structures indomains II and V of 23S ribosomal RNA. Molecular Microbiology 31 (2):623-631. 23S 2058 A to G/U Ery-R, Tyl-R, Lin-R Brachyspira Karlsson, M.,C. Fellstrom, M. U. Heldtander, hyodysenteriae K. E. Johansson, and A.Franklin. 1999. Genetic basis of macrolide and lincosamide resistance inBrachyspira (Serpulina) hyodysenteriae. FEMS Microbiol. Lett. 172:255-260. 23S 2058 A to G Ery-R, Lin-R Chlamydomonas Harris, E. H., B. D.Burkhart, N. W. Gillham, reinhardtii chl. and J. E. Boynton. 1989.Antibiotic resistance mutations in the chloroplast 16S and 23S rRNAgenes of Chlamydomonas reinhardtii: correlation of genetic and physicalmaps of the chloroplast genome. Genetics. 23S 2058 A to G Ery-R, Lin-REscherichia coli Douthwaite, S. 1992. Functional interactions within 23SrRNA involving the peptidyltransferase center. J. Bacteriol. 174:1333-1338. Vester, B., and R. A. Garrett. 1987. A plasmid-coded andsite- directed mutation in Escherichia coli 23S RNA that confers 23S2058 A to U MLSB-R Escherichia coli Sigmund, C. D., M. Ettayebi, and E.A. Morgan. 1984. Antibiotic resistance mutations in 16S and 23Sribosomal RNA genes of Escherichia coli. Nucl Acids Res. 12: 4653-4663.23S 2058 A to C Clr-R Helicobacter Stone, G. G., D. Shortridge, R. K.Flamm, J. Versalovic, pylori J. Beyer, K. Idler, L. Zulawinski, and S.K. Tanaka. 1996. Identification of a 23S rRNA gene mutation inclarithromycin- resistant Helicobacter pylori. Helicobacter. 1: 227-228.23S 2058 A to C Mac-R, Lin-R Helicobacter Occhialini, A., M. Urdaci, F.Doucet- pylori Populaire, C. M. Bébéar, H. Lamouliatte, and F. Mégraud.1997. Macrolide resistance in Helicobacter pylori: rapid detection ofpoint mutations and assays of macrolide binding to ribosomes.Antimicrob. Agents Chemothe 23S 2058 A to C MLSB-R Helicobacter Wang,G., and D. E. Taylor. 1998. Site- pylori specific mutations in the 23SrRNA gene of Helicobacter pylori confer two types of resistance tomacrolide-lincosamide- streptogramin B antibiotics. Antimicrob. AgentsChemother. 42: 1952-1958. 23S 2058 A to C Cla-R HelicobacterDebets-Ossenkopp, Y. J., A. B. Brinkman, pylori E. J. Kuipers, C. M.Vandenbroucke- Grauls, and J. G. Kusters. 1998. Explaining the bias inthe 23S rRNA gene mutations associated with clarithromycin resistance inclinical isolates of Helicobacter pylori. Antimi 23S 2058 A to G Cla-RHelicobacter Versalovic, J., D. Shortridge, K. Kibler, M. V. Griffy,pylori J. Beyer, R. K. Flamm, S. K. Tanaka, D. Y. Graham, and M. F. Go.1996. Mutations in 23S rRNA are associated with clarithromycinresistance in Helicobacter pylori. Antimicrob. Agents Chemother. 40: 423S 2058 A to G Mac-R, Lin-R Helicobacter Occhialini, A., M. Urdaci, F.Doucet- pylori Populaire, C. M. Bébéar, H. Lamouliatte, and F. Mégraud.1997. Macrolide resistance in Helicobacter pylori: rapid detection ofpoint mutations and assays of macrolide binding to ribosomes.Antimicrob. Agents Chemothe 23S 2058 A to G MLSB-R Helicobacter Wang,G., and D. E. Taylor. 1998. Site- pylori specific mutations in the 23SrRNA gene of Helicobacter pylori confer two types of resistance tomacrolide-lincosamide- streptogramin B antibiotics. Antimicrob. AgentsChemother. 42: 1952-1958. 23S 2058 A to G Cla-R HelicobacterDebets-Ossenkopp, Y. J., A. B. Brinkman, pylori E. J. Kuipers, C. M.Vandenbroucke- Grauls, and J. G. Kusters. 1998. Explaining the bias inthe 23S rRNA gene mutations associated with clarithromycin resistance inclinical isolates of Helicobacter pylori. Antimi 23S 2058 A to U MLSB-RHelicobacter Wang, G., and D. E. Taylor. 1998. Site- pylori specificmutations in the 23S rRNA gene of Helicobacter pylori confer two typesof resistance to macrolide-lincosamide- streptogramin B antibiotics.Antimicrob. Agents Chemother. 42: 1952-1958. 23S 2058 A to U Cla-RHelicobacter Debets-Ossenkopp, Y. J., A. B. Brinkman, pylori E. J.Kuipers, C. M. Vandenbroucke- Grauls, and J. G. Kusters. 1998.Explaining the bias in the 23S rRNA gene mutations associated withclarithromycin resistance in clinical isolates of Helicobacter pylori.Antimi 23S 2058 A to G Clr-R Mycobacterium Wallace, R. J., Jr., A.Meier, B. A. Brown, Y. Zhang, abscessus P. Sander, G. O. Onyi, and E. C.Böttger. 1996. Genetic basis for clarithromycin resistance amoungisolates of Mycobacterium chelonae and Mycobacterium abscessus.Antimicrob. Agents Chemother. 40: 1676 23S 2058 A to C/G/U Clr-RMycobacterium Nash, K. A., and C. B. Inderlied. 1995. avium Geneticbasis of macrolide resistance in Mycobacterium avium isolated frompatients with disseminated disease. Antimicrob. Agents Chemother. 39:2625-2630. 23S 2058 A to C/G/U Clr-R Mycobacterium Nash, K. A., and C.B. Inderlied. 1995. avium Genetic basis of macrolide resistance inMycobacterium avium isolated from patients with disseminated disease.Antimicorb. Agents Chemother. 39: 2625-2630. 23S 2058 A to C/G Clr-RMycobacterium Wallace, R. J., Jr., A. Meier, B. A. Brown, Y. Zhang,chelonae P. Sander, G. O. Onyi, and E. C. Böttger. 1996. Genetic basisfor clarithromycin resistance among isolates of Mycobacterium chelonaeand Mycobacterium abscessus. Antimicrob. Agents Chemother. 40: 1676 23S2058 A to C/G/U Clr-R Mycobacterium Meier, A., P. Kirschner, B.Springer, V. A. Steingrube, intracellulare B. A. Brown, R. J. Wallace,Jr., and E. C. Böttger. 1994. Identification of mutations in 23S rRNAgene of clarithromycin-resistant Mycobacterium intracellulare.Antimicrob. Agents Chemother. 38: 38 23S 2058 A to U Clr-R MycobacteriumBurman, W. J., B. L. Stone, B. A. Brown, R. J. Wallace, kansasii Jr.,and E. C. Böttger. 1998. AIDS-related Mycobacterium kansasii infectionwith initial resistance to clarithromycin. Diagn. Microbiol. Infect.Dis. 31: 369-371. 23S 2058 A to G Clr-R Mycobacterium Sander, P., T.Prammananan, A. Meier, K. Frischkorn, smegmatis and E. C. Böttger. 1997.The role of ribosomal RNAs in macrolide resistance. Mol. Microbiol. 26:469-480. 23S 2058 A to G Ery-HR, Spi-MR, Mycoplasma Lucier, T. S., K.Heitzman, S. K. Liu, and P. C. Hu. Tyl-S, Lin-HR pneumoniae 1995.Transition mutations in the 23S rRNA of erythromycin-resistant isolatesof Mycoplasma pneumoniae. Antimicrob. Agents Chemother. 39: 2770-2773.23S 2058 A to G MLSB-R Propionibacteria Ross, J. I., E. A. Eady, J. H.Cove, C. E. Jones, A. H. Ratyal, Y. W. Miller, S. Vyakrnam, and W. J.Cunliffe. 1997. Clinical resistance to erythromycin and clindamycin incutaneous propionibacteria isolated from acne patients is associatedwith mutatio 23S 2058 A to G MLSB-R Streptococcus Tait-Kamradt, A., T.Davies, M. Cronan, M. R. Jacobs, pneumoniae P. C. Appelbaum, and J.Sutcliffe. 2000. Mutations in 23S rRNA and L4 ribosomal protein accountfor resistance in Pneumococcal strains selected in vitro by macrolidepassage. Antimicrobial Agents and 23S 2058 A to G MLSB-R StreptomycesPernodet, J. L., F. Boccard, M. T. Alegre, M. H. Blondelet- ambofaciensRouault, and M. Guerineau. 1988. Resistance to macrolides, lincosamidesand streptogramin type B antibiotics due to a mutation in an rRNA operonof Streptomyces ambofaciens. EMBO J. 7: 277-282. 23S 2058 A to G Ery-RSaccharomyces Sor, F., and H. Fukuhara. 1982. cerevisiae mit.Identification of two erythromycin resistance mutations in themitochondrial gene coding for the large ribosomal RNA in yeast. NucleicAcids Res. 10: 6571-6577. 23S 2058 A to G Ery-R Treponema Stamm, L. V.,and H. L. Bergen. 2000. A pallidum point mutation associated withbacterial macrolide resistance is present in both 23S rRNA genes of anerythromycin-resistant Treponema pallidum clinical isolate [letter].Antimicrob Agents Chemother. 44: 806-807. 23S 2059 “A to G”Clarithomycin Helecobacter Versalovic, J., Shortridge, D., Kibler, K.,resistance. pylori Griffy, M. V., Beyer, J., Flamm, R. K., Tanaka, S.K., Graham, D. Y., and Go, M. F. (1996) Antimicrob. Agents andChemother. 40, 477-480. 23S 2059 “A to G” Lincomycin Tobacco Cseplö, A.,Etzold, T., Schell, J., and resistance. chloroplasts Schreier, P. H.(1988) Mol. Genet. 214, 295-299. 23S 2059 “A to C” Conferred E. coliHansen LH, Mauvais P, Douthwaite S. 1999. resistance to the Themacrolide-kelotide antibiotic binding site MLS drugs and is formed bystructures in domains II and V chloramphenicol. of 23S ribosomal RNA.Molecular Microbiology 31 (2): 623-631. 23S 2059 “A to G” Like A2059C E.coli Hansen LH, Mauvais P, Douthwaite S. 1999. The macrolide-kelotideantibiotic binding site is formed by structures in domains II and V of23S ribosomal RNA. Molecular Microbiology 31 (2): 623-631. 23S 2059 “Ato U” Like A2059C E. coli Hansen LH, Mauvais P, Douthwaite S. 1999. Themacrolide-kelotide antibiotic binding site is formed by structures indomains II and V of 23S ribosomal RNA. Molecular Microbiology 31 (2):623-631. 23S 2059 A to C Mac-R, Lin-R, SB-S Helicobacter Wang, G., andD. E. Taylor. 1998. Site- pylori specific mutations in the 23S rRNA geneof Helicobacter pylori confer two types of resistance tomacrolide-lincosamide- streptogramin B antibiotics. Antimicrob. AgentsChemother. 42: 1952-1958. 23S 2059 A to C Clr-R HelicobacterDebets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers, pylori C. M.Vandenbroucke-Grauls, and J. G. Kusters. 1998. Explaining the bias inthe 23S rRNA gene mutations associated with clarithromycin resistance inclinical isolates of Helicobacter pylori. Antimi 23S 2059 A to G Clr-RHelicobacter Versalovic, J., D. Shortridge, K. Kibler, M. V. Griffy,pylori J. Beyer, R. K. Flamm, S. K. Tanaka, D. Y. Graham, and M. F. Go.1996. Mutations in 23S rRNA are associated with clarithromycinresistance in Helicobacter pylori. Antimicrob. Agents Chemother. 40: 423S 2059 A to G Mac-R, Lin-R Helicobacter Occhialini, A., M. Urdaci, F.Doucet- pylori Populaire, C. M. Bébéar, H. Lamouliatte, and F. Mégraud.1997. Macrolide resistance in Helicobacter pylori: rapid detection ofpoint mutations and assays of macrolide binding to ribosomes.Antimicrob. Agents Chemothe 23S 2059 A to G Mac-R, Lin-R, HelicobacterWang, G., and D. E. Taylor. 1998. Site- SB-S pylori specific mutationsin the 23S rRNA gene of Helicobacter pylori confer two types ofresistance to macrolide-lincosamide- streptogramin B antibiotics.Antimicrob. Agents Chemother. 42: 1952-1958. 23S 2059 A to G Cla-RHelicobacter Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,pylori C. M. Vandenbroucke-Grauls, and J. G. Kusters. 1998. Explainingthe bias in the 23S rRNA gene mutations associated with clarithromycinresistance in clinical isolates of Helicobacter pylori. Antimi 23S 2059A to C/G Clr-R Mycobacterium Wallace, R. J., Jr., A. Meier, B. A. Brown,Y. Zhang, abscessus P. Sander, G. O. Onyi, and E. C. Böttger. 1996.Genetic basis for clarithromycin resistance among isolates ofMycobacterium chelonae and Mycobacterium abscessus. Antimicrob. AgentsChemother. 40: 1676 23S 2059 A to G Clr-R Mycobacterium Wallace, R. J.,Jr., A. Meier, B. A. Brown, Y. Zhang, chelonae P. Sander, G. O. Onyi,and E. C. Böttger. 1996. Genetic basis for clarithromycin resistanceamong isolates of Mycobacterium chelonae and Mycobacterium abscessus.Antimicrob. Agents Chemother. 40: 1676 23S 2059 A to C Clr/Azm-RMycobacterium Meier, A., P. Kirschner, B. Springer, V. A. Steingrube,intracellulare B. A. Brown, R. J. Wallace, Jr., and E. C. Böttger. 1994.Identification of mutations in 23S rRNA gene of clarithromycin-resistantMycobacterium intracellulare. Antimicrob. Agents Chemother. 38: 38 23S2059 A to C Clr/Azm-R Mycobacterium Meier, A., P. Kirschner, B.Springer, V. A. Steingrube, avium B. A. Brown, R. J. Wallace, Jr., andE. C. Böttger. 1994. Identification of mutations in 23S rRNA gene ofclarithromycin-resistant Mycobacterium intracellulare. Antimicrob.Agents Chemother. 38: 38 23S 2059 A to G Clr-R Mycobacterium Sander, P.,T. Prammananan, A. Meier, K. Frischkorn, smegmatis and E. C. Böttger.1997. The role of ribosomal RNAs in macrolide resistance. Mol.Microbiol. 26: 469-480. 23S 2059 A to G Ery-MR, Spi-HR, MycoplasmaLucier, T. S., K. Heitzman, S. K. Liu, and P. C. Hu. Tyl-LR, Lin-MRpneumoniae 1995. Transition mutations in the 23S rRNA oferythromycin-resistant isolates of Mycoplasma pneumoniae. Antimicrob.Agents Chemother. 39: 2770-2773. 23S 2059 A to G Mac-R StreptococcusTait-Kamradt, A., T. Davies, M. Cronan, M. R. Jacobs, pneumoniae P. C.Appelbaum, and J. Sutcliffe. 2000. Mutations in 23S rRNA and L4ribosomal protein account for resistance in Pneumococcal strainsselected in vitro by macrolide passage. Antimicrobial Agents and 23S2059 A to G Mac-HR, Lin-LR Propionibacteria Ross, J. I., E. A. Eady, J.H. Cove, C. E. Jones, A. H. Ratyal, Y. W. Miller, S. Vyakrnam, and W. J.Cunliffe. 1997. Clinical resistance to erythromycin and clindamycin incutaneous propionibacteria isolated from acne patients is associatedwith mutatio 23S 2060 “A to C” E. coli Vester, B. and Garrett, R. A.(1988) EMBO J. 7, 3577-3587. 23S 2061 “G to A” Chloramphenicol Ratmitochondria Vester, B. and Garrett, R. A. (1988) EMBO resistance J. 7,3577-3587. 23S 2062 “A to C” Chloramphenicol Halobacterium Mankin, A. S.and Garrett, R. A. (1991) J. resistance. halobium Bacteriol. 173,3559-3563. 23S 2251 “G to A” Dominant lethal; E. coli Green, R., Samaha,R., and Noller, H. Abolished both (1997). J. Mol. Biol. 266, 40-50.binding of tRNA and peptidyl transferase activity. 23S 2251 “G to A”Dominant lethal; E. coli Gregory, S. T. and Dahlberg, A. E. subunitassociation (unpublished). defect. 23S 2251 “G to C” Dominant lethal E.coli Gregory, S. T. and Dahlberg, A. E. subunit association(unpublished). defect. 23S 2251 “G to U” Dominant lethal E. coliGregory, S. T. and Dahlberg, A. E. subunit association (unpublished).defect. 23S 2251 “G to U” Dominant lethal; E. coli Green, R., Samaha,R., and Noller, H. Abolished both (1997). J. Mol. Biol. 266, 40-50.binding of tRNA and peptidyl transferase activity. 23S 2251 “G to A”Dominant lethal; E. coli Gregory ST, Dahlberg AE, 1999. Mutationsimpairs peptidyl in the Conserved P Loop Perturb the transferaseactivity; Conformation of Two Structural Elements induces DMS in thePeptidyl Transferase Center of 23 S reactivity; induces Ribosomal RNA.J. Mol. Biol. 285: 1475-1483. kethoxal reactivity in G2238, G2409,G2410, G2529, and G2532; enhances CMCT reactivity in G2238; induceskethoxal and CMCT reactivity in G2269 and G2271; induces CMCT reactivityin U2272 and U2408; enhances kethoxal reactivity in G2253. 23S 2252 “Gto A” Less than 5% of E. coli Porse, B. T., Thi-Ngoc, H. P., andGarrett, R. A. control level (1996) J. Mol. Biol. 264: 472-486. peptidyltransferase activity. 23S 2252 “G to C” Less than 5% of E. coli Porse,B. T., Thi-Ngoc, H. P., and Garrett, R. A. control level (1996) J. Mol.Biol. 264: 472-486. peptidyl transferase activity. 23S 2252 “G to U”Less than 5% of E. coli Porse, B. T., Thi-Ngoc, H. P., and Garrett, R.A. control level (1996) J. Mol. Biol. 264: 472-486. peptidyl transferaseactivity. 23S 2252 “G to C” Reduced rate of E. coli 1. Lieberman, K. R.and Dahlberg, A. E. peptidyl transferase (1994) J. Biol. Chem. 269,16163-16169. bond formation in 2. Samaha, R. R., Green R., and Noller,H. F. vitro; severely (1995) Nature 377, 309-314. 3. detrimental to cellO'Connor, M., Brunelli, C. A., Firpo, M. A., growth. Double Gregory, S.T., Lieberman, K. R., Lodmell, J. S., mutation Moine, H., Van Ryk, D.I., and (G2252C/G2253C). Dahlberg, A. E. (1995) Cell Biol. 73, 859-868.23S 2252 “G to A” Severely detrimental E. coli 1. Gregory, S. T.,Lieberman, K. R., and to cell growth; Dahlberg, A. E. (1994) NucleicAcids Res. promoted 22, 279-284. 2. Lieberman, K. R. and frameshiftingand Dahlberg, A. E. (1994) J. Biol. Chem. 269, readthrough of16163-16169. nonsense codons. 23S 2252 “G to C” Severely detrimental E.coli 1. Gregory, S. T., Lieberman, K. R., and to cell growth; Dahlberg,A. E. (1994) Nucleic Acids Res. promoted 22, 279-284. 2. Lieberman, K.R. and frameshifting and Dahlberg, A. E. (1994) J. Biol. Chem. 269,readthrough of 16163-16169. nonsense codons. 23S 2252 “G to U” Severelydetrimental E. coli 1. Gregory, S. T., Lieberman, K. R., and to cellgrowth; Dahlberg, A. E. (1994) Nucleic Acids Res. promoted 22, 279-284.2. Lieberman, K. R. and frameshifting and Dahlberg, A. E. (1994) J.Biol. Chem. 269, readthrough of 16163-16169. nonsense codons. 23S 2252“G to A” Dominant lethal; E. coli Gregory ST, Dahlberg AE, 1999. impairspeptidyl Mutations in the Conserved P Loop transferase activity; Perturbthe Conformation of Two induces DMS Structural Elements in the Peptidylreactivity; induces Transferase Center of 23 S Ribosomal kethoxalreactivity in RNA. J. Mol. Biol. 285: 1475-1483. G2238, G2409, G2410,G2529, and G2532; enhances CMCT reactivity in G2238; induces kethoxaland CMCT reactivity in G2269 and G2271; induces CMCT reactivity in U2272and U2408; enhances kethoxal reactivity in G2253. 23S 2252 “G to A”Interfere with the E. coli Bocchetta M, Xiong L, Mankin AS. 1998.building of peptidyl- 23S rRNA positions essential for tRNA tRNA to Psite of 50S binding in ribosomal functional sites. Proc. subunit. Natl.Acad. Sci. 95: 3525-3530. 23S 2252 “G to C” Interferes with the E. coliBocchetta M, Xiong L, Mankin AS. 1998. binding of peptidyl- 23S rRNApositions essential for tRNA tRNA to P site of 50S binding in ribosomalfunctional sites. Proc. subunit Natl. Acad. Sci. 95: 3525-3530. 23S 2252“G to U” Interferes with the E. coli Bocchetta M, Xiong L, Mankin AS.1998. binding of peptidyl- 23S rRNA positions essential for tRNA tRNA toP site of 50S binding in ribosomal functional sites. Proc. subunit Natl.Acad. Sci. 95: 3525-3530. 23S 2252 “G to U” Dominant lethal; E. coliNitta I, Ueda T, Watanabe K. 1998. suppressed AcPhe- Possibleinvolvement of Escherichia coli Phe formation; 23S ribosomal RNA inpeptide bond suppressed peptide formation. RNA 4: 257-267. bondformation. c 23S 2253 “G to C” 42% control level E. coli Porse, B. T.,Thi-Ngoc, H. P., and Garrett, R. A. peptidyl transferase (1996) J. Mol.Biol. 264: 472-486. activity. 23S 2253 “G to C” Slow growth rate. E.coli Gregory, S. T., Lieberman, K. R., and Dahlberg, A. E. (1994)Nucleic Acids Res. 22, 279-284. 23S 2253 “G to C” Promoted E. coli 1.Lieberman, K. R. and Dahlberg, A. E. frameshifting and (1994) J. Biol.Chem. 269, 16163-16169. readthrough of 2. Samaha, R. R., Green R., andNoller, H. F. nonsense codons. (1995) Nature 377, 309-314. 3. O'Connor,M., Brunelli, C. A., Firpo, M. A., Gregory, S. T., Lieberman, K. R.,Lodmell, J. S., Moine, H., Van Ryk, D. I., and Dahlberg, A. E. (1995)Cell Biol. 73, 859-868. 23S 2253 “G to A” Promoted E. coli 1. Lieberman,K. R. and Dahlberg, A. E. frameshifting and (1994) J. Biol. Chem. 269,16163-16169. readthrough of 2. Samaha, R. R., Green R., and Noller, H.F. nonsense codons. (1995) Nature 377, 309-314. 3. O'Connor, M.,Brunelli, C. A., Firpo, M. A., Gregory, S. T., Lieberman, K. R.,Lodmell, J. S., Moine, H., Van Ryk, D. I., and Dahlberg, A. E. (1995)Cell Biol. 73, 859-868. 23S 2253 “G to U” Promoted E. coli 1. Lieberman,K. R. and Dahlberg, A. E. frameshifting and (1994) J. Biol. Chem. 269,16163-16169. readthrough of 2. Samaha, R. R., Green R., and Noller, H.F. nonsense codons. (1995) Nature 377, 309-314. 3. O'Connor, M.,Brunelli, C. A., Firpo, M. A., Gregory, S. T., Lieberman, K. R.,Lodmell, J. S., Moline, H., Van Ryk, D. I., and Dahlberg, A. E. (1995)Cell Biol. 73, 859-868. 23S 2253 “G to A” 19% of control level E. coliPorse, B. T., Thi-Ngoc, H. P., and Garrett, R. A. peptidyl transferase(1996) J. Mol. Biol. 264: 472-486. activity. 23S 2253 “G to C” Severelydetrimental E. coli 1. Lieberman, K. R. and Dahlberg, A. E. to cellgrowth; (1994) J. Biol. Chem. 269, 16163-16169. reduced rate of 2.Samaha, R. R., Green R., and Noller, H. F. peptide bond (1995) Nature377, 309-314. 3. formation in vitro. O'Connor, M., Brunelli, C. A.,Firpo, M. A., Double mutations Gregory, S. T., Lieberman, K. R.,Lodmell, J. S., (C2253C/2252C). Moine, H., Van Ryk, D. I., and Dahlberg,A. E. (1995) Cell Biol. 73, 859-868. 23S 2253 “G to U” Less than 5%control E. coli Porse, B. T., Thi-Ngoc, H. P., and Garrett, R. A. levelpeptidyl (1996) J. Mol. Biol. 264: 472-486. transferase activity. 23S2253 “G to A” Induced DMS E. coli Gregory ST, Dahlberg AE, 1999.reactivity; enhanced Mutations in the Conserved P Loop CMCT reactivityin Perturb the conformation of Two G2238; induced Structural Elements inthe Peptidyl kethoxal and CMCT Transferase center of 23 S Ribosomalreactivity in G2269 RNA. J. Mol. Biol. 285: 1475-1483. and G2271;induced CMCT reactivity in U2272; induced kethoxal reactivity in G2409and G2410. 23S 2253 “G to C” Induced DMS E. coli Gregory ST, DahlbergAE, 1999. reactivity; enhanced Mutations in the Conserved P Loop CMCTreactivity in Perturb the Conformation of Two G2238; induced StructuralElements in the Peptidyl kethoxal and CMCT Transferase Center of 23 SRibosomal reactivity in G2269 RNA. J. Mol. Biol. 285: 1475-1483 andG2271; induced CMCT reactivity in U2272; induced kethoxal reactivity inG2409 and G2410 23S 2438 “U to A” Amecetin resistance HalobacteriumLeviev, I. G., Rodriguez-Fonseca, C., and reduced growth halobium Phan,H., Garrett, R. A., Heliek, G., Noller, H. F., rate. and Mankin, A. S(1994) EMBO J. 13, 1682-1686 23S 2438 “U to C” Amicetin resistance.Halobacterium Leviev, I. G., Rodriguez-Fonseca, C., halobium Phan, H.,Garrett, R. A., Heliek, G., Noller, H. F., and Mankin, A. S (1994) EMBOJ. 13, 1682-1686. 23S 2438 “U to G” Unstable in presence HalobacteriumLeviev, I. G., Rodriguez-Fonseca, C., or absence of halobium Phan, H.,Garrett, R. A., Heliek, G., Noller, H. F., amicetin and Mankin, A. S(1994) EMBO J. 13, 1682-1686. 23S 2447 “G to A” Chloramphenicol YeastDujon, B. (1980) Cell 20, 185-197. resistance. mitochondria 23S 2447 “Gto C” Anisomycin Halobacterium Hummel, H. and Böck, A. (1987)resistance. halobium Biochimie 69, 857-861. 23S 2450 “A to C” Lethal. E.coli Vester, B. and Garrett, R. A. (1988) EMBO J. 7, 3577-3587. 23S 2451“A to U” Chloramphenicol Mouse Kearsey, S. E. and Craig, I. W. (1981)resistance. mitochondria Nature (London) 290: 607-608. 23S 2451 “A to G”Like A2451G E. coli Bocchetta M, Xiong L, Mankin AS. 1998. 23S rRNApositions essential for tRNA binding in ribosomal functional sites.Proc. Natl. Acad. Sci. 95: 3525-3530. 23S 2451 “A to C” Like A2451G E.coli Bocchetta M, Xiong L, Mankin AS. 1998. 23S rRNA positions essentialfor tRNA binding in ribosomal functional sites. Proc. Natl. Acad. Sci.95: 3525-3530. 23S 2452 “C to A” Chloramphenicol Human Blanc, H., WrightC. T., Bibb M. J., Wallace D. C., resistance. mitochondria and ClaytonD. A. (1981) Proc. Natl. Acad. Sci. USA 78, 3789-3793. 23S 2452 “C to U”Animosycin resistance. Halobacterium Hummel, H. and Böck, A. (1987)Biochimie 69, 857-861. 23S 2452 “C to U” Animosycin resistanceTetrahymena Sweeney, R., Yao, C. H., and Yao, M. C. thermophilia (1991)Genetics 127: 327-334. 23S 2452 “C to U” Chloramphenicol HalobacteriumMankin, A. S. and Garrett, R. A. (1991) J. resistance. halobiumBacteriol. 173: 3559-3563. 23S 2452 “C to U” Chloramphenicol MouseSlott, E. F., Shade R. O., and Lansman, R. A. resistance mitochondria(1983) Mol. Cell. Biol. 3, 1694-1702. 23S 2452 “C to U” Low levelsparsomycin Halobacterium Tan, G. T., DeBlasio, A., and Mankin, A. S.resistance halobium (1996) J. Mol. Biol. 261, 222-230. 23S 2452 C to UCbm-R, Lin-R Sulfolobus Aagaard, C., H. Phan, S. Trevisanato, andacidocaldarius R. A. Garrett. 1994. A spontaneous point mutation in thesingle 23S rRNA gene of the thermophilic arachaeon Sulfolobusacidocaldarius confers multiple drug resistance. J. Bacteriol. 176:7744-7747. 23S 2453 “A to C” Anisomycin resistance Halobacterium Hummel,H. and Böck, A. (1987) Biochimie halobium 69, 857-861. 23S 2492 “U to A”Frameshift E. coli O'Connor, M. and Dahlberg, A. E. (1996) suppressors.Nucleic Acids Res. 24, 2701-2705. 23S 2492 “U to C” Frameshift E. coliO'Connor, M. and Dahlberg, A. E. (1996) suppressors. Nucleic Acids Res.24, 2701-2705. 23S 2493 “del U” (With A2058G and E. coli O'Connor, M.and Dahlberg, A. E. (1996) erythromycin) Lethal Nucleic Acids Res. 24,2701-2705. growth effects. Frameshift suppressors. 23S 2493 “U to A”(With A2058G and E. coli 1. Porse, B. T. and Garrett, R. A. (1995) J.erythromycin) Lethal Mol. Biol. 249, 1-10. 2. O'Connor, M., growtheffects. Brunelli, C. A., Firpo, M. A., Gregory, S. T., Frameshiftsuppressors Lieberman, K. R., Lodmell, J. S., Moine, H., Van Ryk, D. I.,and Dahlberg, A. E. (1995) Biochem. Cell Biology 73, 859-868. 23S 2493“U to C” (With A2058G and E. coli 1. Porse, B. T. and Garrett, R. A.(1995) J. erythromycin) Lethal Mol. Biol. 249, 1-10. 2. O'Connor, M.,growth effects. Brunelli, C. A., Firpo, M. A., Gregory, S. T.,Frameshift suppressors Lieberman, K. R., Lodmell, J. S., Moine, H., VanRyk, D. I., and Dahlberg, A. E. (1995) Biochem. Cell Biology 73,859-868. 3.O'Connor, M. and Dahlberg, A. E. (1996) Nucleic Acids Res.24, 2701-2705 23S 2493 “U to C” (With A2058G and E. coli O'Connor, M.and Dahlberg, A. E. (1996) erythromycin) Lethal Nucleic Acids Res. 24,2701-2705 growth effects. Frameshift suppressors 23S 2493 “U to G” (WithA2058G and E. coli O'Connor, M. and Dahlberg, A. E. (1996) erythromycin)Lethal Nucleic Acids Res. 24, 2701-2705 growth effects. Frameshiftsuppressors 23S 2493 “U to A” (With A2058G and E. coli O'Connor, M. andDahlberg, A. E. (1996) erythromycin) Lethal Nucleic Acids Res. 24,2701-2705 growth effects. Frameshift suppressors 23S 2493 “U to C”Increased misreading. E. coli O'Connor, M. and Dahlberg, A. E. (1996)Double mutation Nucleic Acids Res. 24, 2701-2705 (U2493C/G2458A) 23S2493 “U to C” Increased misreading. E. coli O'Connor, M. and Dahlberg,A. E. (1996) Double mutation Nucleic Acids Res. 24, 2701-2705(U2493C/G2458C) 23S 2497 “A to G” (With A2058G and E. coli Porse, B. T.and Garrett, R. A. (1995) J. erythromycin) Reduced Mol. Biol. 249, 1-10.growth rate. 23S 2499 “C to U” Sparsomycin Halobacterium Tan, G. T.,DeBlasio, A. and Mankin, A. S. resistance halobium (1996) J. Mol. Biol.261, 222-230 23S 2500 U2500A/C2501A Inhibits binding of 1A E. coli PorseBT, Garrett RA. 1999. Sites of streptogramin B, Interaction ofStreptogramin A and B antibiotic pristinamycin Antibiotics in thePeptidyl Transferase 1A on peptidyl Loop of 23 S rRNA and the Synergismof transferase loop their Inhibitory Mechanisms. J. Mol. Biol. causinginhibition of 286: 375-387. peptide elongation. c 23S 2500 U2500A/C2501GLike U2500A/C2501A. c E. coli Porse BT, Garrett RA. 1999. Sites ofInteraction of Streptogramin A and B Antibiotics in the PeptidylTransferase Loop of 23 S rRNA and the Synergism of their InhibitoryMechanisms. J. Mol. Biol. 286: 375-387. 23S 2500 U2500A/C2501U LikeU2500A/C2501A. c E. coli Porse BT, Garrett RA. 1999. Sites ofInteraction of Streptogramin A and B Antibiotics in the PeptidylTransferase Loop of 23 S rRNA and the Synergism of their InhibitoryMechanisms. J. Mol. Biol. 286: 375-387. 23S 2500 U2500G/C2501A LikeU2500A/C2501A. c E. coli Porse BT, Garrett RA. 1999. Sites ofInteraction of Streptogramin A and B Antibiotics in the PeptidylTransferase Loop of 23 S rRNA and the Synergism of their InhibitoryMechanisms. J. Mol. Biol. 286: 375-387. 23S 2500 U2500G/C2501G LikeU2500A/C2501A. c E. coli Porse BT, Garrett RA. 1999. Sites ofInteraction of Streptogramin A and B Antibiotics in the PeptidylTransferase Loop of 23 S rRNA and the Synergism of their InhibitoryMechanisms. J. Mol. Biol. 286: 375-387. 23S 2500 U2500G/C2501U LikeU2500A/C2501A. c E. coli Porse BT, Garrett RA. 1999. Sites ofInteraction of Streptogramin A and B Antibiotics in the PeptidylTransferase Loop of 23 S rRNA and the Synergism of their InhibitoryMechanisms. J. Mol. Biol. 286: 375-387. 23S 2500 U2500C/C2501A LikeU2500A/C2501A. c E. coli Porse BT, Garrett RA. 1999. Sites ofInteraction of Streptogramin A and B Antibiotics in the PeptidylTransferase Loop of 23 S rRNA and the Synergism of their InhibitoryMechanisms. J. Mol. Biol. 286: 375-387. 23S 2500 U2500C/C2501G LikeU2500A/C2501A. c. E. coli Porse BT, Garrett RA. 1999. Sites ofInteraction of Streptogramin A and B Antibiotics in the PeptidylTransferase Loop of 23 S rRNA and the Synergism of their InhibitoryMechanisms. J. Mol. Biol. 286: 375-387. 23S 2500 U2500C/C2501A LikeU2500A/C2501A. c E. coli Porse BT, Garrett RA. 1999. Sites ofInteraction of Streptogramin A and B Antibiotics in the PeptidylTransferase Loop of 23 S rRNA and the Synergism of their InhibitoryMechanisms. J. Mol. Biol. 286: 375-387. 23S 2502 “G to A” Decreasedgrowth rate E. coli Vester, B. and Garrett, R. A. (1988) EMBO J. 7,3577-3587 23S 2503 “A to C” Chloramphenicol Yeast Dujon, B. (1980) Cell20, 185-197 resistance mitochondria 23S 2503 “A to C” Decreased growthrate; E. coli Porse, B. T. and Garrett, R. A. (1995) J. CAMr Mol. Biol.249, 1-10. 23S 2503 “A to G” (With A2058G and E. coli Porse, B. T. andGarrett, R. A. (1995) J. erythromycin) Slow Mol. Biol. 249, 1-10. growthrate. CAMr 23S 2504 “U to A” Increased readthrough E. coli O'Connor, M.,Brunelli, C. A., Firpo, M. A., of stop codons and Gregory, S. T.,Lieberman, K. R., Lodmell, J. S., frameshifting; lethal Moine, H., VanRyk, D. I., and Dahlberg, A. E. (1995) Biochem. Cell Biology 73,859-868. 23S 2504 “U to C” Increased readthrough E. coli O'Connor, M.,Brunelli, C. A., Firpo, M. A., of stop codons and Gregory, S. T.,Lieberman, K. R., Lodmell, J. S., frameshifting; lethal Moine, H., VanRyk, D. I., and Dahlberg, A. E. (1995) Biochem. Cell Biology 73,859-868. 23S 2504 “U to C” Chloramphenicol Mouse Blanc, H., Wright, C.T., Bibb, M. J., resistance mitochondria Wallace, D. C., and Clayton, D.A. (1981) Proc. Natl. Acad. Sci. USA 78, 3789-3793 23S 2504 “U to C”Chloramphenicol Human Kearsey, S. E., and Craig, I. W. (1981) resistancemitochondria Nature (London) 290, 607-608 23S 2505 “G to A” 14% activityof 70S E. coli Porse, B. T., Thi-Ngoc, H. P. and Garrett, R. A.ribosomes (1996) J. Mol. Biol. 264, 472-486 23S 2505 “G to C” (WithA1067U and E. coli 1. Saarma, U. and Remme, J. (1992) thiostrepton)Temperature Nucleic Acids Res. 23, 2396-2403. 2. sensitive growth. aSaarma, U., Lewicki, B. T. U., Margus, T., Hypersensitivity to CAM;Nigul, S., and Remme, J. (1993) “The increased sensitivity of inTranslational Apparatus: Structure, vitro translation. Slight Function,Regulation and Evolution” 163-172. increase in sensitivity tolincomycin. b No effect on translational accuracy. 23S 2505 “G to C”Excluded from 70S E. coli Porse, B. T. Thi-Ngoc, H. P. and Garrett, R.A. ribosomes; 17% activity of (1996) J. Mol. Biol. 264, 472-486 70Sribosomes 23S 2505 “G to U” <5% activity of 70S E. coli Porse, B. T.,Thi-Ngoc, H. P. and Garrett, R. A. ribosomes (1996) J. Mol. Biol. 264,472-486 23S 2505 “G to A” Conferred resistance to E. coli Hansen LH,Mauvais P, Douthwaite S. the MLS drugs and 1999. The macrolide-kelotideantibiotic chloramphenicol. binding site is formed by structures indomains II and V of 23S ribosomal RNA. Molecular Microbiology 31 (2):623-631. 23S 2505 “G to C” Like G2505A. E. coli Hansen LH, Mauvais P,Douthwaite S. 1999. The macrolide-kelotide antibiotic binding site isformed by structures in domains II and V of 23S ribosomal RNA. MolecularMicrobiology 31 (2): 623-631. 23S 2505 “G to U” Like G2505A E. coliHansen LH, Mauvais P, Douthwaite S. 1999. The macrolide-kelotideantibiotic binding site is formed by structures in domains II and V of23S ribosomal RNA. Molecular Microbiology 31 (2): 623-631. 23S 2506 “Uto A” Dominant lethal; 5% E. coli Porse, B. T., Thi-Ngoc, H. P. andGarrett, R. A. activity of 70S ribosomes (1996) J. Mol. Biol. 264,472-486 23S 2508 “A to U” Eryr, Cdr, Cms; abolishes E. coli 1. Sigmund,C. D., Ettayebi, M., and methylation of 23S rRNA Morgan, E. A. (1984)Nucleic Acids Res. by ErmE. 12, 4653-4663. 2. Vannuffel, P., DiGiambattista, M., and Cocito, C. (1992) J. Biol. Chem. 267, 16114-16120.3. Douthwaite, S. and Aagaard, C. (1993) J. Mol. Biol. 232, 725-731. 4.Vester, B., Hansen, L. H., and Douthwaite, S. (1995) RNA 1, 501-509. 23S2508 “G to U” Control level peptidyl E. coli 1. Porse, B. T. andGarrett, R. A. (1995) J. transferase activity Mol. Biol. 249, 1-10. 2.Porse, B. T., Thi- Ngoc, H. P. and Garrett, R. A. (1996) J. Mol. Biol.264, 472-486 23S 2514 “U to C” Control level peptidyl E. coli Porse, B.T. and Garrett, R. A. (1995) J. transferase activity Mol. Biol. 249,1-10. 23S 2516 “A to U” Control level peptidyl E. coli Porse, B. T. andGarrett, R. A. (1995) J. transferase activity Mol. Biol. 249, 1-10. 23S2528 “U to A” (With A2058G and E. coli Porse, B. T. and Garrett, R. A.(1995) J. erythromycin) Slow growth Mol. Biol. 249, 1-10. rate. Controllevel peptidyl transferase activity 23S 2528 “U to C” Control levelpeptidyl E. coli Porse, B. T. and Garrett, R. A. (1995) J. transferaseactivity Mol. Biol. 249, 1-10. 23S 2530 “A to G” (With A2058G and E.coli Porse, B. T. and Garrett, R. A. (1995) J. erythromycin) Slow growthMol. Biol. 249, 1-10. rate. 23S 2546 “U to C” Control level peptidyl E.coli Porse, B. T. and Garrett, R. A. (1995) J. transferase activity.Mol. Biol. 249, 1-10. 23S 2550 “G to A” (With A2058G and E. coli Porse,B. T. and Garrett, R. A. (1995) J. erythromycin) Slow growth Mol. Biol.249, 1-10. rate. 23S 2552 “U to A” (With A2058G and E. coli Porse, B. T.and Garrett, R. A. (1995) J. erythromycin) Slow growth Mol. Biol. 249,1-10. rate. 23S 2555 “U to A” Stimulates readthrough of E. coli 1.O'Connor, M. and Dalhberg, A. E. stop codons and (1993) Proc. Natl.Acad. Sci. USA 90, frameshifting; U to A is 9214-9218 2. O'Connor, M.,Brunelli, C. A., trpE91 frameshift Firpo, M. A., Gregory, S. T.,suppressor; viable in low Lieberman, K. R., Lodmell, J. S., Moine, H.,copy number plasmids, Van Ryk, D. I., and Dahlberg, A. E. but lethalwhen expressed (1995) Biochem. Cell Biology 73, 859-868. constitutivelyfrom lambda pL promoter 23S 2555 “U to C” (With A2058G and E. coliPorse, B. T. and Garrett, R. A. (1995) J. erythromycin) Slow growth Mol.Biol. 249, 1-10. rate. Control level peptidyl transferase activity 23S2555 “U to C” no effect E. coli O'Connor, M. and Dalhberg, A. E. (1993)Proc. Natl. Acad. Sci. USA 90, 9214-9218 23S 2557 “G to A” (With A2058Gand E. coli Porse, B. T. and Garrett, R. A. (1995) J. Mol. erythromycin)Slow growth Biol. 249, 1-10. rate. Intermediate decrease in peptidyltransferase activity. 23S 2565 “A to U” (With A2058G and E. coli Porse,B. T. and Garrett, R. A. (1995) J. Mol. erythromycin) Slow growth Biol.249, 1-10. rate. Very low peptidyl transferase activity. 23S 2580 “U toC” (With A2058G and E. coli Porse, B. T. and Garrett, R. A. (1995) J.Mol. erythromycin) Lethal growth Biol. 249, 1-10. effects. No peptidyltransferase activity. 23S 2581 “G to A” Dominant lethal inhibition of E.coli 1. Spahn, C., Reeme, J., Schafer, M. and puromycin in reactionNierhaus, K. (1996) J. Biol. Chem. 271, 32849-32856 2. Spahn, C., Reeme,J., Schafer, M. and Nierhaus, K. (1996) J. Biol. Chem. 271, 32857-3286223S 2584 “U to A” Deleterious; 20% activity of E. coli Porse, B. T.,Thi-Ngoc, H. P. and Garrett, R. A. 70S ribosomes (1996) J. Mol. Biol.264, 472-486 23S 2584 “U to C” Deleterious; 20% activity of E. coliPorse, B. T., Thi-Ngoc, H. P. and Garrett, R. A. 70S ribosomes (1996) J.Mol. Biol. 264, 472-486 23S 2584 “U to G” (With A2058G and E. coliPorse, B. T. and Garrett, R. A. (1995) J. Mol. erythromycin) Lethalgrowth Biol. 249, 1-10. effects. No peptidyl transferase activity. 23S2589 “A to G” (With A2058G and E. coli Porse, B. T. and Garrett, R. A.(1995) J. erythromycin) Slow growth Mol. Biol. 249, 1-10. rate. Strongreduction in peptidyl transferase activity. 23S 2602 A2602C/C2501AInhibits binding of 1A E. coli Porse BT, Kirillov SV, Awayez MJ,streptogramin B, antibiotic Garrett RA. 1999. UV-induced pristinamycin1A on modifications in the peptidyl peptidyl transferase looptransferase loop of 23S rRNA causing inhibition of dependent on bindingof the peptide elongation. c streptogramin B antibiotic pristinamycinIA. RNA 5: 585-595. 23S 2602 A2602C/C2501U Like A2602C/C2501A. c E. coliPorse BT, Kirillov SV, Awayez MJ, Garrett RA. 1999. UV-inducedmodifications in the peptidyl transferase loop of 23S rRNA dependent onbinding of the streptogramin B antibiotic pristinamycin IA. RNA 5:585-595. 23S 2602 A2602C/C2501G Like A2602C/C2501A. c E. coli Porse BT,Kirillov SV, Awayez MJ, Garrett RA. 1999. UV-induced modifications inthe peptidyl transferase loop of 23S rRNA dependent on binding of thestreptogramin B antibiotic pristinamycin IA. RNA 5: 585-595. 23S 2602A2602U/C2501A Like A2602C/C2501A. c E. coli Porse BT, Kirillov SV,Awayez MJ, Garrett RA. 1999. UV-induced modifications in the peptidyltransferase loop of 23S rRNA dependent on binding of the streptogramin Bantibiotic pristinamycin IA. RNA 5: 585-595. 23S 2602 A2602U/C2501U LikeA2602C/C2501A. c E. coli Porse BT, Kirillov SV, Awayez MJ, Garrett RA.1999. UV-induced modifications in the peptidyl transferase loop of 23SrRNA dependent on binding of the streptogramin B antibioticpristinamycin IA. RNA 5: 585-595. 23S 2602 A2602U/C2501G LikeA2602C/C2501A. c E. coli Porse BT, Kirillov SV, Awayez MJ, Garrett RA.1999. UV-induced modifications in the peptidyl transferase loop of 23SrRNA dependent on binding of the streptogramin B antibioticpristinamycin IA. RNA 5: 585-595. 23S 2602 A2602G/C2501A LikeA2602C/C2501A. c E. coli Porse BT, Kirillov SV, Awayez MJ, Garrett RA.1999. UV-induced modifications in the peptidyl transferase loop of 23SrRNA dependent on binding of the streptogramin B antibioticpristinamycin IA. RNA 5: 585-595. 23S 2602 A2602G/C2501U LikeA2602C/C2501A. c E. coli Porse BT, Kirillov SV, Awayez MJ, Garrett RA.1999. UV-induced modifications in the peptidyl transferase loop of 23SrRNA dependent on binding of the streptogramin B antibioticpristinamycin IA. RNA 5: 585-595. 23S 2602 A2602G/C2501G LikeA2602C/C2501A. c E. coli Porse BT, Kirillov SV, Awayez MJ, Garrett RA.1999. UV-induced modifications in the peptidyl transferase loop of 23SrRNA dependent on binding of the streptogramin B antibioticpristinamycin IA. RNA 5: 585-595. 23S 2611 “C to G” Erythromycin andChlamydomonas Gauthier, A., Turmel, M. and Lemieux, C. spiramycinresistance reinhardtii (1988) Mol. Gen. Genet. 214, 192-197. 23S 2611 “Cto G” Erythromycin and Yeast Sor, F. and Fukahara, H. (1984) spiramycinresistance mitochondria Nucleic Acids Res. 12, 8313-8318. 23S 2611 “C toU” Eryr and low level Chlamydomonas Harris, E. H., Burkhart, B. D.,Gillham, N. W. lincomycin and clindamycin reinhardtii and Boynton, J. E.(1989) resistance Genetics 123, 281-292 23S 2611 “C to G” Eryr and lowlevel Chlamydomonas Harris, E. H., Burkhart, B. D., Gillham, N. W.lincomycin and clindamycin reinhardtii and Boynton, J. E. (1989)resistance Genetics 123, 281-292 23S 2611 “C to U” Slightly Eryr;reduced E. coli Vester, B., Hansen, L. H., and methylation DoubleDouthwaite, S. (1995) RNA 1, 501-509 mutation (C2611U/ G2057A) 23S 2611C to G Ery-R, Spi-LR Chlamydomonas Gauthier, A., M. Turmel, and C.Lemieux. moewusii 1988. Mapping of chloroplast chl. mutations conferringresistance to antibiotics in Chlamydomonas: evidence for a novel site ofstreptomycin resistance in the small subunit rRNA. Mol. Gen. Genet. 214:192-197. 23S 2611 C to G/U Ery-R, Lin-MR Chlamydomonas Harris, E. H., B.D. Burkhart, N. W. Gillham, reinhardtii and J. E. Boynton. 1989. chl.Antibiotic resistance mutations in the chloroplast 16S and 23S rRNAgenes of Chlamydomonas reinhardtii: correlation of genetic and physicalmaps of the chloroplast genome. Genetics. 23S 2611 C to U Ery-R, Spi-S,Tyl-S, Lin-S Escherichia Vannuffel, P., M. Di Giambattista, E. A.Morgan, coli and C. Cocito. 1992. Identification of a single base changein ribosomal RNA leading to erythromycin resistance. J. Biol. Chem. 267:8377-8382. 23S 2611 C to A/G Mac-R, SB-S Streprococcus Tait-Kamradt, A.,T. Davies, M. Cronan, pneumoniae M. R. Jacobs, P. C. Appelbaum, and J.Sutcliffe. 2000. Mutations in 23S rRNA and L4 ribosomal protein accountfor resistance in Pneumococcal strains selected in vitro by macrolidepassage. Antimicrobial Agents and 23S 2611 C to G Ery-R, Spi-RSaccharomyces Sor, F., and H. Fukuhara. 1984. cerevisiae Erythromycinand spiramycin mit. resistance mutations of yeast mitochondria: natureof the rib2 locus in the large ribosomal RNA gene. Nucleic Acids Res.12: 8313-8318. 23S 2611 C to U Ery-S, Spi-R Saccharomyces Sor, F., andH. Fukuhara. 1984. cerevisiae Erythromycin and spiramycin mit.resistance mutations of yeast mitochondria: nature of the rib2 locus inthe large ribosomal RNA gene. Nucleic Acids Res. 12: 8313-8318. 23S 2661“G to C” Decreased misreading; E. coli 1. Tapprich, W. E. and Dalhberg,A. E. streptomycin dependent (1990) EMBO J. 9, 2649-2655 2. Tapio, S.when expressed with Smr, and Isaksson, L. A. (1991) Eur. J.hyperaccurate S12 Biochem. 202, 981-984 3. Melancon, P., mutation.Tapprich, W. and Brakier-Gingras, L. (1992) J. Bacteriol. 174, 7896-79014. Bilgin, N. and Ehrenberg, M. (1994) J. Mol. Biol. 235, 813-824 5.O'Connor, M., Brunelli, C. A., Firpo, M. A., Gregory, S. T., Lieberman,K. R., Lodmell, J. S., Moine, H., Van Ryk, D. I., and Dahlberg, A. E.(1995) Biochem. Cell Biology 73, 859-868. 23S 2661 “C to A” Like C2661E. coli Munishkin A, Wool IG. 1997. The ribosome-in-pieces: Binding ofelongation factor EF-G to oligoribonucleotides that mimic thesarcin/ricin and thiostrepton domains of 23S ribosomal RNA. Proc. Natl.Acad. Sci. 94: 12280-12284. 23S 2661 “C to G” Like C2661 E. coliMunishkin A, Wool IG. 1997. The ribosome-in-pieces: Binding ofelongation factor EF-G to oligoribonucleotides that mimic thesarcin/ricin and thiostrepton domains of 23S ribosomal RNA. Proc. Natl.Acad. Sci. 94: 12280-12284. 23S 2661 “C to U” Like C2661 E. coliMunishkin A, Wool IG. 1997. The ribosome-in-pieces: Binding ofelongation factor EF-G to oligoribonucleotides that mimic thesarcin/ricin and thiostrepton domains of 23S ribosomal RNA. Proc. Natl.Acad. Sci. 94: 12280-12284. 23S 2666 “C to G” Increased stop codon E.coli O'Connor, M. and Dalhberg, A. E. (1996) readthrough and NucleicAcids Res. 24, 2701-2705 frameshifting. a Double mutation(C2666G/A2654C) 23S 2666 “C to G” Minor increase in stop E. coliO'Connor, M. and Dalhberg, A. E. (1996) codon readthrough and NucleicAcids Res. 24, 2701-2705 frameshifting. a Double mutation(C2666G/A2654U) 23S 2666 “C to U” Minor increase in stop E. coliO'Connor, M. and Dalhberg, A. E. (1996) codon readthrough and NucleicAcids Res. 24, 2701-2705 frameshifting. a Double mutation(C2666U/A2654C) 23S 2666 “C to U” Significant increase in stop E. coliO'Connor, M. and Dalhberg, A. E. (1996) codon readthrough and NucleicAcids Res. 24, 2701-2705 frameshifting. a Double mutation(C2666U/A2654G) 23S 2666 “C to U” Minor increase in stop E. coliO'Connor, M. and Dalhberg, A. E. (1996) codon readthrough and NucleicAcids Res. 24, 2701-2705 frameshifting. a Double mutation(C2666U/A2654U)

TABLE 4 Effect of resistance on % Change in Resistance PathogenRecomended Etest Mechanism fluorescence Fluorescenc MRSA MethicillinResistant SA Staphylococcus aureus Reduced affinity of PBP2 towardspenicillins; Reduction of Penicillin binding > to ≈ 80% nosocomial,Multi-drug-resistant (clindamycin, reduction gentamicin, FQ); ContainSCCmec type I, II, or III; Usually PVL-negative; Virulent (esp. skin andlung) CA-MRSA Community acquired MRSA; StaphylococcusMulti-drug-resistant (clindamycin, gentamicin, FQ); Reduction ofPenecillin > to ≈ 80% aureus Usually only resistant to pen, ox ± eryth ±FQs; Usually binding. Maintaims binding reduction of produce PVL,especially in the US; In general the capacity for clindamycin andpenicillin, organisms remain susceptible to clindamycin and totrimethoprim clindamycin trimethoprim sulfa. That's different from anosocomial trimethoprim pathogen which is usually resistant to one ofthese binding antibiotics. PVL-MRSA Panton-Valentine StaphylococcusHighly abundant toxin, scausing septic shock; large — — leukocidin -aureus complications MRSA BORSA Border.line Staphylococcus OxacillinReduction of Penecillin > to ≈ 80% oxacillin resistant aureus binding.reduction of SA penicillin ORSA Oxacillin Staphylococcus OxacillinReduction of Penecillin > to ≈ 80% resistant SA aureus binding.reduction of penicillin VRSA Vancomycin Staphylococcus Vancomycin;Teicoplanin Modified phenotypic features, however, include slowerIncreased binding of resistant SA aureus growth rates, a thickened cellwall, and increased levels Vancomycin of PBP2 and PBP2′ (although thedegree of cross-linking within the thick cell wall seems to be reduced)[58]. Vancomycin resistant strains also seem to have a greater abilityto absorb the antibiotic from the outside medium, which may be aconsequence of the greater availability of stem peptides in the thickcell wall. In addition, the VRS Vancomycin Staphylococci Vancomycinincreased amounts of two PBPs may compete with the Increased bindingof >50% resistant Staph (CNS) antibiotic for the stem peptidesubstrates, thus Vancomycin >50% aggravating the resistance profile VISAVancomycin Staphylococcus Vancomycin Increased binding of 20-50%intermediary SA aureus Vancomycin 20-50% hVISA hetero-(resistant)Staphylococcus Vancomycin very rare, but can be susceptible tomethicillin and Binding of penicillin and Vancomycin aureus resistant tovancomycin vancomycin VRE Vancomycin Enterococci Vancomycin; TeicoplaninReduced affinity to Van by 3 orders of magnitude Reduction ofvancomycin >80% resistant binding reduction ESBL ExtendedEnterobacteriaceae Overproduction of β-Lactamases, inhibited byclavulanic Increased binding of >80% Spectrum β- acid clavulanic acidincrease Lactamase ESBL Extended Pseudomonas Ceftazidime Overproductionof β-Lactamases, inhibited by clavulanic Increased binding of >80%Spectrum β- spp. acid clavulanic acid increase Lactamase ESBL ExtendedAcinetobacter Ceftazidime Overproduction of β-Lactamases, inhibited byclavulanic Increased binding of >80% Spectrum β- acid clavulanic acidincrease Lactamase ESBL Extended BCC Ceftazidime Overproduction ofβ-Lactamases, inhibited by clavulanic Increased binding of >80% Spectrumβ- acid clavulanic acid increase ESBL Extended StenotrophomonasCeftazidime Overproduction of β-Lactamases, inhibited by clavulanicIncreased binding of >80% Spectrum β- maltophilia acid clavulanic acidincrease Lactamase MBL Metalo-β- Imipenem — — Lactamase MLS macrolideOne mechanism is called MLS, macrolide lincosamide Reduced bindingof >50% lincosamide streptogramin. And in this situation there is analteration macrolides and Ketolides streptogramin in a target-bindingsite at the 23-ribosomal RNA level. resulting in a point mutation ormethylation of 23SRNA resulting in reduced binding of macrolides (alsoKetolides); organisms with an efflux mechanis will bind macrolides underFISH conditions. They are also sensitive to clindamycin DRSPdrug-resistant Modify DR is reported for beta-lactams, macrolides,Reduction of macrolides >50% S. pneumoniae clavulanic acid,chloramphenicol, and sulfonamides & single gene detection for effluxpump HLAR High level Enterococci Gentamycin; Streptomycin Reduction inStreptomycin >80% Aminoglycoside binding reduction Resistance inEnterococci

REFERENCES

-   1. Chambers, H. F. (1997). Methicillin resistance in staphylococci:    molecular and biochemical basis and clinical implications. Clinical    Microbiology Reviews 10,781-791.-   2. Swenson, J. M., Williams, P., Killgore, G. et al. (2001).    Performance of eight methods, including two new methods for    detection of oxacillin resistance in a challenge set of    Staphylococcus aureus organisms. Journal of Clinical Microbiology    39, 3785-8-   3. Van Leeuwen, W. B., Van Pelt, C., Luijendijk, A. et al. (1999).    Rapid detection of methicillin resistance in Staphylococcus aureus    isolates by the MRSA-screen latex agglutination test. Journal of    Clinical Microbiology 37, 3029-30.-   4. Louie, L., Majury, A., Goodfellow, J. et al. (2001). Evaluation    of a latex agglutination test (MRSA-Screen) for detection of    oxacillin resistance in coagulase-negative staphylococci. Journal of    Clinical Microbiology 39, 4149-51.-   5. Skov, R., Smyth, R., Clausen, M. et al. (2003). Evaluation of    cefoxitin 30 μg disc on Iso-Sensitest agar for detection of    methicillin-resistant Staphylococcus aureus. Journal of    Antimicrobial Chemotherapy 52, 204-7-   6. Felten, A., Grandry, B., Lagrange, P. H. et al. (2002).    Evaluation of three techniques for detection of low-level    methicillin-resistant S. aureus (MRSA): a disc diffusion method with    cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen    latex agglutination test. Journal of Clinical Microbiology 40,    2766-71.-   7. Cauwelier, B., Gordts, B., Descheemaecker, P. et al. (2004).    Evaluation of a disk diffusion method with cefoxitin (30 μg) for    detection of methicillin-resistant Staphylococcus aureus. European    Journal of Clinical Microbiology and Infectious Diseases 23, 389-92.-   8. Kluytmans, J., Van Griethuysen, A., Willemse, P. et al. (2002).    Performance of CHROM agar selective medium and oxacillin resistance    screening agar base for identifying Staphylococcus aureus and    detecting methicillin resistance. Journal of Clinical Microbiology    40, 2480-2.-   9. Louie, L., Matsumura, S. O., Choi, E. et al. (2000). Evaluation    of three rapid methods for detection of methicillin resistance in S.    aureus. Journal of Clinical Microbiology 38, 2170-3.-   10. National Committee for Clinical Laboratory Standards. (2003).    Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria    that Grow Aerobically: Approved Standards M7-A6 and M100-S13. NCCLS,    Wayne, Pa., USA.

1. Method for the detection of an antibiotic resistance in apredetermined micro-organism in a biological sample, comprising thesteps: (a) providing a labelled antibiotic, (b) contacting the labelledantibiotic with a biological sample comprising the micro-organism underconditions which allow binding of the labeled antibiotic to its bindingsite in the micro-organism, (c) detecting the labelled antibiotic in themicro-organism, and (d) identifying a micro-organism in which the amountof detectable label is altered with respect to the amount of detectablelabel in the predetermined micro-organism in its non-resistant form,wherein micro-organisms identified in step (d) are microorganismresistant against the antibiotic.
 2. The method according to claim 1,wherein the antibiotic is selected from the group consisting ofaminoglycosides, carbacephems, carbapenems, cephalosporins,glycopeptides, macrolides, monobactams, beta-lactam antibiotics,quinolones, bacitracin, sulfonamides, tetracyclines, streptogramines,chloramphenicol, clindamycin, and lincosamide.
 3. The method accordingto claim 1, wherein the antibiotic is labelled by a luminescentlabelling group, in particular by a fluorescent labelling group.
 4. Themethod according to claim 1, wherein the binding site is located in thecell lumen, in the cytoplasm, in the cell wall, or/and in a secretedprotein.
 5. The method according to claim 1, wherein the antibiotic is abeta-lactam antibiotic.
 6. The method according to claim 5, wherein thebeta-lactam antibiotic binds to the PBP2 binding protein.
 7. The methodaccording to claim 1, wherein the label in step (c) is detected viaepifluorescence microscopy, flow cytometry, laser scanning devices, timeresolved fluorometry, luminescence detection, isotope detection, hyperspectral imaging scanner, Surface Plasmon Resonance or anotherevanscesence based reading technology.
 8. The method according to claim1, wherein the predetermined microorganism is identified in thebiological sample.
 9. The method according to claim 8, wherein thepredetermined microorganism is identified by a labelled nucleic acidcapable of specifically hybridising with a nucleic acid in themicro-organism under in-situ conditions.
 10. Method according to claim8, wherein the predetermined microorganism is identified by FISH. 11.The method according to claim 8, wherein the predeterminedmicro-organism is identified in step (c).
 12. The method according toclaim 8, wherein the identification of the micro-organism and thedetection of the labelled antibiotic in the micro-organism are runconcurrently.
 13. The method according to claim 8, wherein themicroorganism is detected via epifluorescence microscopy, flowcytometry, laser scanning devices, time resolved fluorometry,luminescence detection, isotope detection, hyper spectral imagingscanner, Surface Plasmon Resonance or another evanscesence based readingtechnology.
 14. The method according to claim 1, wherein micro-5organism is selected from the group consisting of bacteria, yeasts andmoulds, in particular from Gram positive and Gram negative bacteria. 15.The method of claim 1, wherein the micro-organism is a Gram positivebacterium which is perforated by the formulation Gram PositivePerforation Buffer in Table
 2. 16. The method according to claim 1,wherein the micro-organism is a yeasts or a mould, which is perforatedby the formulation Yeast Perforation Buffer in Table
 2. 17. The methodaccording to claim 9, wherein a Methicillin Resistant Staphylococcusaureus (MRSA) is identified by in-situ hybridisation simultaneously withthe detection of the expression of the Penicillin Binding Protein 2 bybinding a labelled β-Lactam antibiotic.
 18. The method according toclaim 9, wherein a Methicillin Resistant Staphylococcus aureus (MRSA) isidentified by in-situ hybridisation simultaneously with thedifferentiation between nosocomial and community acquired MRSA via therespective ability to bind labelled5 Clindamycin or Trimethoprim sulfa.19. The method according to claim 9, wherein a Vancomycin ResistantStaphylococcus aureus (VRSA) is identified by in-situ hybridisationsimultaneously with the ability to bind labelled Vancomycin.
 20. Themethod according to claim 9, wherein a Vancomycin ResistantStaphylococcus (VRS) is identified by in-situ hybridisationsimultaneously with the ability to bind labelled Vancomycin.
 21. Themethod according to claim 9, wherein a Vancomycin Resistant Enterococci(VRE) is identified by in-situ hybridisation simultaneously with theability to bind labelled Vancomycin.
 22. The method according to claim8, wherein a resistance towards β-lactam antibiotics due to thesecretion of β-lactamase (ESBL) is detected by the revealing of thepresence of said enzyme by the binding of labelled clavulanic acidtogether with the identification of Gram negative micro-organisms. 23.The method according to claim 8, wherein a resistance towards β-lactamantibiotics due to the secretion of metalo-β-lactamases (MBL) isdetected by the revealing of the presence of said enzyme by the bindingof labelled imipenem together with the identification of Gram negativemicro-organisms.
 24. The method according to claim 8, wherein aresistance to macrolides, lincosamide and streptogramin (MLS) isdetected via the binding of labelled erythromycin or/and Clindamycintogether with the identification of Streptococci.
 25. The methodaccording to claim 8, wherein a drug resistant Streptococcus pneumoniae(DRSP) is identified with FISH together with the respective resistancetowards beta-lactams and macrolides.
 26. The method according to claim8, wherein high level Aminoglycoside resistant Enterococci (HLAR) aredetected via FISH and labelled Gentamycin.
 27. The method of claim 1which is a diagnostic method.
 28. A kit suitable for detecting anantibiotic resistance in a predetermined micro-organism, comprising (a)a labelled antibiotic, and (b) optionally a probe suitable foridentification of the micro-organism in the biological sample.
 29. Useof a labelled antibiotic for the detection of an antibiotic resistancein a predetermined micro-organism in a biological sample.